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1. set label color def lc color str LabelColorSetScoped color ScratchSubset str set screen center def center str ViewerCenterSetScoped ScratchSubset str show objects def show str OEPySetVisible ScratchSubsetList str True hide objects def hide str OEPySetVisible ScratchSubsetList str False In this manner any of the AFITT scripting commands which take a scope argument can be easily ex panded to take a selection string CHAPTER SIXTEEN Preferences Every user has his or her own individual preferences with regards to how molecules grids and surfaces should look and how applications should behave For this reason a Preferences dialog is available which allows customization of the application to the user s preference An snapshot of the Preferences dialog can be seen in Figure 16 1 Preferences General Molecule Input Output Molecules Grids O pen Files From Surfaces 3D Display Current Directory Last Directory Modeling Ligand Browsing Slideshow Speed ms Miscellaneous C Allow multiple application instances C Compress grid contour information in mini state files C Write empty SD data Y Warn before executing large operations 20 Number of operations Restore Apply Save Cancel Figure 16 1 Preferences On the left hand side of the dialog is column containing preference categories These categories include General Molecules
2. Also may use the pre defined sets a sch to match side chain atoms and a Sbackbone or a Sba to match backbone atoms e an Atom number This integer property matches the atom number Example an 5 e r Residue name This string property matches the residue name Example r ALA See also the pre defined sets at section 15 5 e rn Residue number This integer property matches the residue number Example rn 1 50 e ch Chain This string property matches the chain The chain should be a single letter Example ch A e model Model number This integer property matches atoms with the given PDB style model number i e for different NMR models Example mode1 2 e altloc Alternate Location indicator This string property matches atoms with the given PDB style AlternateLocation property The string should be a single letter Example alt loc A 124 Chapter 15 Query language icode Insertion code This string property matches atoms with the the given PDB style insertion code Example icode B occ Occupancy This floating point property matches atoms with the the given occupancy prop erty Example occ gt 0 5 b B factor This floating point property matches atoms with the the given crystallographic tem perature B factor Example b gt 50 0 q Partial charge This floating point property matches atoms with the the given partial charge Example q gt 0 O Formal charge This f
3. List Window Name cox2_good sdf gz 1 1 cox2 1 3 cox2 1 4 cox2 1 5 cox2 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 1 Ory onn pone E EEE E x2 0000000000000 a E E E E 8 E 1 30 cox2 1 31 cox2 1 32 cox2 iH 1 33 cox2 4 1 34 cox2 1 35 cox2 1 36 cox2 Figure 12 4 Changing multiple objects simultaneously 12 5 1 Creating Lists New lists can be created in several ways The simplest way is the New List submenu in the File menu A number of list creation mechanisms are available and include using the AND of two or more lists intersection the OR of two or more lists union the XOR of two lists union intersection or from the currently Marked set of objects The AND list contains only those objects which are in all of the source lists The OR list contains all of the objects contained in its individual source lists The XOR list contains the objects which are in only one of the source lists but not both Finally the Marked list creates a new list which contains all of the currently Marked objects The second way to create a new list is by right clicking on an individual list and selecting the Subset submenu from the popup menu This menu contains four entries By Marked By Query By Not Marked and By Not Query The By Marked option creates a new list containing all of the Marked objects from within the list The
4. Restore Selecting one of these options will crop away the unselected portion of the surface crop away the selected portion of the surface or restore the surface to its original state respectively Examples of cropped surfaces can be seen in Figure 5 5 5 1 User Interaction 39 In the bottom row there is one button adjacent to a slider and a numeric display The button is a toggle that controls whether or not unselected atoms and bonds are shown based on their distance to the selected set The cutoff distance is controlled by the adjacent slider and the actual value in Angstroms can be seen in the numeric display This state of this button is ignored when a surface is selected When a vertex or a line of vertices is selected the slider can be used to flood out from the original selected set to include many more adjacent vertices The different types of surface selections can be seen in Figure 5 4 a Single vertex selected b Line of vertices selected c Region of vertices selected Figure 5 4 Collection of different possible selection states on a surface a Unselected region removed b Selected region removed Figure 5 5 Different styles of cropping a surface Style The style pane of the Style Control is divided into two distinct areas as can be seen in Figure 5 6 The area above the dividing line contains a row of four buttons which control the display style of the various different types of objects that can be
5. Lastly at the bottom of the Process Editor is a Python Handler field which allows for specification 13 1 Processes 115 MM input Editor Filename Source Molecule s oeb O Grid phi Python E B Default Input Figure 13 3 Process Input Editor of a Python function to be run when the process is completed This allows for handling of output not normally handled by AFITT or for output which is not specified on the command line 13 1 1 Adding Input The Input Editor allows the user to specify a mechanism by which an input file is provided to the process to be run At the top of the editor are two mutually exclusive options the first is the Temporary check box which indicates that the file to be used will be a temporary file which will be removed after the process has completed The other option is a field which allows specification of a specific filename to be used In both cases the user has a choice as to how this file will be populated saving molecules saving grids or running a specified Python function which will handle the population of the file If molecules or grids are chosen a file format to save these in must be specified in the pull down box next to the selected option If a Python function is specified to populate the input file the desired filename can be obtained by using the keyword FILE in the function specification This keyword will be substituted with the real filenam
6. cox2_good sdf gz E 1 3 cox2 E 1 4 cox2 1 5 cox2 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 10 cox2 1 16 cox2 1 17 cox2 1 18 cox2 1 19 cox2 1 20 cox2 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 1 29 cox2 1 30 cox2 1 31 cox2 1 32 cox2 1 33 cox2 1 34 cox2 1 35 cox2 1 36 cox2 Oy nn pone Figure 12 1 List window 123 Views The List Window provides three different views onto the loaded data Which view is shown is controlled by the three buttons at the bottom of the List Window The first view is a traditional hierarchical view described above and seen in Figure 12 1 The second view is a flat view see Figure 12 2 a in which the list hierarchy has been removed but all the contents are shown in the same original order The third view is a spreadsheet view see Figure 12 2 b which shows only those objects that appear within the associated spreadsheet as specified by the drop down box next to its button Furthermore the order of the objects in this view parallels the current sorting order in the application spreadsheet For more details about the spreadsheet please see Chapter 11 12 4 List Columns Every object in the List Window may populate one of the several columns described in the introduction Some columns are simply informative such as the unique identifier while others are functional as well as informative Clicking on an object s name will m
7. e pkey This unsigned integer property limits the match for the part of the query it appears in to match only objects whose parent has the given key Example pkey 100000001 15 5 Macros Pre defined sets In addition to explicitly naming residues and atoms AFITT defines several macros which may be used in query strings These macros are prefixed with a dollar sign Macros are generally used with the residue name i e r property although a few are used with the atom name i e a property The definitions for these sets are largely borrowed from RasMol e r Saliphatic ALA GLY LEU VAL ILE PRO e r Shydroxyl SER THR TYR e r Ssulfur CYS MET e r Saromatic TYR HIS TRP PHE e r Scharged ASP GLU ARG LYS e r Samide GLN ASN e r Shydrophobic ALA GLY LEU VAL ILE PRO MET PHE TRP e r polar SER THR CYS TYR HIS ASP GLU ASN GLN ARG LYS e r Sneutral ALA GLY LEU VAL ILE PRO SER THR CYS MET PHE TYR TRP ASN GLN e r Sacidic ASP GLU e r Sbasic ARG LYS e r Ssmall1l ALA GLY SER 126 Chapter 15 Query language e r Smedium VAL PRO THR CYS ASP ASN e r large ILE MET PHE TYR HIS TRP GLU GLN ARG LYS e r cyclic HIS PRO TYR TRP PHE e r Sdna ADE A GUA G CYT C THY T URA U e r Saa all amino acids e r at ADE A THY T URA U e r cg CYT C GUA G e r purine ADE A GUA G e r Spyrimidine CYT C THY T URA U e r Sw
8. Browse Figure 4 6 Capturing a screenshot 4 5 3 Python A complete history of the application s operations can be written out as a Python script This script can be exported by selecting the Script history option in the Export submenu in the File menu 4 6 Drag and Drop A powerful drag and drop interface is provided for passing molecules images of molecules and molec ular data between applications Multiple mechanisms of transfering data via the drag and drop interface are supported e Dragging a list of filenames into the List Window see Chapter 12 will cause those files to be loaded e Dragging a list of SMILES into the List Window will cause those SMILES to be parsed into new molecules and stored in a new list called Pasted These new molecules will not be assigned 3D coordinates and therefore will not be visible in the 3D display see Chapter 5 e Dragging a list of IUPAC or common chemical names into the List Window will cause those names to be parsed into new molecules using Lexichem and stored in a new list called Pasted Please note this functionality requires a separate license for the Lexichem toolkit available from OpenEye These new molecules will not be assigned 3D coordinates and therefore will not be visible in the 3D display see Chapter 5 4 6 Drag and Drop 33 e Dragging molecules from other molecular visualization programs into the List Window will cause those molecules to be
9. the protein only portion of the coordinates e ligand pdb the ligand only portion of the coordinates These files are suitable both for experimentation and for testing with the supplied automation scripts 133 CHAPTER EIGHTEEN Example Scripts AFITT 1 3 ships with an example scripts directory as an example of AFITT s scripting capability In cluded in this directory are 134 pdbopen py This script creates a new menu option in the File menu called Open From PDB which allows AFITT to open molecule files directly from the online Protein Data Bank Simply enter the PDB id and if AFITT is able to locate that id in the database it will download the file and load it into AFITT cmds py This script defines some commands which use the AFITT query language to do com mon tasks like centering and coloring atoms or labels dbsmiles py This script demonstrates how to connect AFITT to a database such as a corporate registry system refmac_params py This contains the original refmac parameters The variables in this file can be used as templates to change the default refmac parameters afitt_automate py This script demonstrates how to automatically fit a ligand to density The overall process is to read a mtz file create a map from it find blobs of unfilled density generate conformers for a specified ligand to go into that density fit the conformers to the density There are many opportunities for customizing t
10. tion of High Quality Atomic Charges AM1 BCC Model I Method Journal of Computational Chemistry Vol 21 pp 132 146 2000 Araz Jakalian David B Jack and Christopher I Bayly Fast Efficient Generation of High Quality Atomic Charges AM1 BCC Model II Parameterization and Validation Journal of Computa tional Chemistry Vol 23 pp 1623 1641 2002 http www eyesopen com docs
11. Box File SEAL dl C 5 10 14 The first line indicates the number of atoms The next line is the name and the remaining lines are the atoms and coordinates The coordinates are separate by white space tabs or spaces work just fine and specified in Angstroms For example the results of selecting Add Box on the 1115 ligand located in the examples directory is displayed in Figure 7 7 3 There are two things to notice e Associated with the box is a newly created molecule named Bounding Box e The box has two spheres at either ends these are actually dummy atoms Once a box has been generated Mouse Mode Translate operation can be used to change its size 7 7 4 Use a Neutral pH Model For The Ligand In the Ligand Preferences page a neutral pH model can be selected for use with conformer generation Otherwise the Task Generate Neutral pH menu option will apply a neutral pH model to the currently focused or selected ligand 76 Chapter 7 Ligand Modeling If the neutral pH model is applied to a protein it most likely will generate undesired results for incomplete chains or residues Note Changing the pH model for a ligand or protein is not undoable The molecule will have to be reloaded 7 7 5 SCIiNI Find Active Sites In this case you can use the Generate Negative Image option of the Task menu to attempt to find the active sites of the protein This generates a Shape Contour Negative Image by running a collecti
12. FDELWT Amplitidues for difference map PHIDELWT Phases for difference map Table 19 2 MTZ Column Switches Map Types Fo Fe Fo Fe 2Fo Fe 3Fo 2Fc 5Fo 3Fc 2Fo FeSigmaA Fo FcSigmaA FoFom Fwt Fdelwt 140 Chapter 19 Automation 19 2 6 Filtering on protein distance By default blobs farther away than 4 Angstroms from the protein are not selected for fitting To change this behavior use the distance flag or change the default value of the slider in the Ligand Model window The following example sets a protein distance of 10 0A bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit boxes lig pdb gz distance 10 0 19 3 Platform Differences 19 3 1 Windows To run automation from windows AFITT must be started from the command line using the supplied bat file If AFITT is installed to the default location afitt automation scripts will be launched as follows C OpenEye AFITT 1 3 0 afitt bat e automate 19 3 2 OSX Due to the way OS X bundles applications to start automation from the command line the bundle must be treated as a directory structure If AFITT is installed into the Applications directory the command line to start automation is as follows Applications afitt cpp Contents MacOS afitt e automate Sadly before the automation begins the environment details are also output CHAPTER TWENTY Mouse Maps 20 1 AFITT Mouse Map Table
13. Marked Visible Choosing any one of these options will create a new molecule which is composed of all the molecules that were either Selected Marked or Visible at the time of the operation 5 9 4 Monitors The ability to display measurement monitors is a useful feature and is well supported Access to these measurement facilities is available from the Mouse popup toolbar on the left hand side of the 3D display There are three buttons which turn on Distance Angle and Torsion measurement respectively a Distance Monitor b Angle Monitor c Torsion Monitor Figure 5 16 Different types of monitors When the mouse is put into one of these modes the measurements are made based on the selected set of atoms In Distance mode the first atom selected is consider the anchor atom Once the anchor atom has been selected a temporary distance monitor will be displayed to any other atom that the mouse passes over Selecting another atom will create a permanent monitor and clear the anchor In Angle mode the first two atoms selected are considered the anchor atoms and a temporary angle monitor will be displayed to any other atom that the mouse passes over Selecting a third atom will create a permanent monitor and clear the anchors In Torsion mode the first three atoms selected are considered the anchor atoms and a temporary torsion monitor will be displayed to any other atom that the mouse passes over Selecting a
14. Other products and software packages referenced in this document are trademarks and registered trade marks of their respective vendors or manufacturers CONTENTS Introduction 1 Installation 2 Z1 Microsoft WiNdOwWS 24 64 ba Rk ke ad ASS e ra YS 2 22 MINE cerda sr WW GG eth ak a ee da ae e Ba SS 2 23 MACOS ea dos a a dae tay ah Uae a ke oe we Sk ad tay es Shas Bie ae OS 3 DA S S dr ack GA es ta ool ws Al ale De we bom Bk Bee Bak a Mand Hel Ge a ALS 3 Getting Started 5 3 1 Why Another Modelling Program 1 2 ee ee 5 3 2 General Concepts es s ui aaa ewe Rw ERE OR OE we we 6 3 3 Object States co cocoa eR ewe DE RH Em Re Be we 6 2 OCODE a nd sados sto da Bl aad dav da Goin LR ay bi da 8 Be A a eee ek aed a tare Ged Sos Gh Ghee ona Ae Gos eed ins 8 360 Undo Red cs sisi o a de a Ow ee ee we A 11 Sal SID Window USAS se w s a e iedh a ee a ee HG ee Bk eo a 12 3 6 Spreadsheet Usage snio s Qc aw Me dom AUSE Sati doar a we el Sard a e 12 39 2D Preview Usage us a seed A AP a ek Gea ie aw Soar ava es 12 LIO HOw toiCenter e so ele a aee a aae AAN Stee do aracs boa Mee Se ws Bae wk ad 13 SoM WAGs 2 wot eee Sete ae haw eo ON beer end brag oe e aa Sr S yer aes eae Oe a ee A 13 3 12 Ligand Task oedi 24 eau bh Soe i ee REE EEE A Oe er Ree 13 SS PO asker ja a Sie a Ae de A at Bede a a ae es SL Aen Gs RA Ge 17 OMA PRG iS 95 72 a eae Bo fae Ao RN 22 File VO 23 Ad Opening Ple ccoo dina a a ae gh E 23 AD Clos
15. Spagna R Watkin D Automatic solution and refinement of crystal structures by means of the package UNIQUE Acta Cryst A Vol 47 pp 373 381 Altomare A Giacovazzo C Ianigro M Moliterni A Rizzi R Peak labelling in electron density maps from powder data the use of crystal chemical information J Appl Cryst Vol 35 pp 21 27 2002 Zwart P Langer G Lamzin V Modelling bound ligands in protein crystal structures Acta Cryst D Vol 60 pp 2230 2239 2004 Oldfield T Creating structure features by data mining the PDB to use as molecular replacement models Acta Cryst D Vol 57 pp 696 705 2001 Perola E Charifson P S Conformational analysis of drug like molecules bound to proteins an extensive study of ligand reorganization upon binding J Med Chem Vol 47 pp 2499 510 2004 Wlodek S Skillman A G Nicholls A Automated ligand placement and refinement with a com bined force field and shape potential Acta Cryst D 2005 Vagin A A Steiner R A Lebedev A A Potterton L McNicholas S Long F and Murshudov G N REFMACS dictionary organization of prior chemical knowledge and guidelines for its use Acta Cryst D Vol 60 pp 2184 2195 2004 151 152 Bibliography 12 13 14 15 16 17 18 19 20 21 Murshudov G N Vagin A A Dodson E J Refinement of Macromolecular structures by Maxi mum l
16. The Refmac Launcher widget is seen in Figure 3 14 MM Refmac Launcher Destination jocuments and Settings brian My Documents afitt_refine Destination Path Method normal y Refinement Type normal or rigid Molecules 6 1428 protein Molecules To Refine 8 1428 ligand Reflection Data 4 1428_prot mtz y Reflection Data MTZ F FP 7 sus EC Reflection Parameters Feehan Cycles Refinement cycles XRay Weight Refinement XRay Weight Extra Arguments Figure 3 14 Refmac Launcher To view the status of the refinement simply switch to the Refinement task This workflow shows the current refinements that are being or have been run and their status When a refinement is complete a prompt will appear and the results can be loaded into AFITT or ignored as desired Note even if refmacS is not installed then AFITT writes the appropriate data dictionary and pdb file and the refmacS scripts to the destination for future use CHAPTER FOUR File 1 O 4 1 Opening Files There are multiple mechanisms by which a file can be loaded in AFITT The simplest method is to select the Open option in the File menu This will launch a file selection dialog which prompts the user for the desired files s which will subsequently be loaded A short list of the most recently opened files can also be found in the File menu under the Recents or Open Recents on Mac OS X submenu Selecting an option in this submenu will load the assoc
17. appropriate drop down box If that function requires additional input such as functions like cos the cursor in the expression editor window will automatically be placed inside the function Functions can be combined with standard math operators such as and If a function requires additional input that is not provided an error will be displayed in the Test section at the bottom of the dialog as seen in Figure 11 4 b Frequently the desired input is contained within another column which can be referenced by selecting the desired column from the third drop down box or simply by typing COL COLUMN NAME as seen in Figure 11 4 c Once the desired expression has been assembled the column can be created and added to the spreadsheet by clicking on the Create button Chapter 11 Spreadsheet Name Description acos x Return the arc cosine measured in radians of x asin x Return the arc sine measured in radians of x atan x Return the arc tangent measured in radians of x atan2 y x Return the arc tangent measured in radians of y x Unlike atan y x the signs of both x and y are considered ceil x Return the ceiling of x as a float This is the smallest integral value gt x cos x Return the cosine of x measured in radians cosh x Return the hyperbolic cosine of x degrees x Converts angle x from radians to degrees e The numeric constant e or 2 7182818284590451 exp x Return e fabs x R
18. lt ri gt K zj z nio vjan mio Figure 8 4 Residue Controls Once the operation mutate append etc has been chosen clicking on the appropriate amino acid code will perform the desired operation The rotamer button selects the highest probability rotamers for the selected residue from the current rotamer library The available rotamer libraries are either the Dunbrack or Richardson rotamer library 14 16 When the rotamer search button is pressed if no density is presently modeled the highest probability 8 6 Bond Specific Operations 85 rotamer is selected If density is present the rotamer that best fits the surrounding density is selected The arrows step through the available choices in the rotamer library 8 6 Bond Specific Operations Bond operations either operate on the selected bond when bond order is being changed or the selected atoms when bonds are being created Break Bond Determine Bonds JEJEJE Sacrifice Fly Double Single l Create Bond Figure 8 5 Bond Controls 1 Break Bonds break the modeled bonds 2 Determine Bonds determine the bond types for the selected residue note that if only part of the residue is selected this determines the types for the whole residue 3 Single bond change the selected bond to a single bond or if no bond exists generate a single bond between the selected atoms 4 Double bond change the selected bond to a double bond or if n
19. passes the filename of the reflection data which to the script It is the user s responsibility therefore to make sure that the file still exists where it did when AFITT read it There are also fields for specifying the number of cycles the xray weight and the column names for the given fields When a data file is chosen AFITT will make its best guess as to the appropriate column names but these may need to be changed this before launching the refinement Finally there is a space for extra arguments These extra arguments are passed onto refmac5 as command line arguments Finally pressing the Launch button will call the given script with the specified parameters Although not necessary the progress of the script can be monitored from the Task Refinement workflow Once refinement is finished AFITT will notify the user that the job is complete and ask if the output file s from the job should be read 9 2 Altering Refmacs Parameters AFITT uses the python scripting language to prepare and launch refmac5 There are two python variables that hold the default refmac5 scripts e REFMAC_RIGID holds the parameters for the rigid refinement e REFMAC_RIGID_PARAMS holds the parameters for the rigid refinement These parameters can be modified by either changing them in the scripting window not recommended loading a python script with the new definitions or placing the new definitions in your startup py file see Chapter 14 2
20. to fit to the density inside the region this is useful when there is very poor density in which blobs can t be found use the boxonly flag bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit boxes lig pdb gz boxpadding boxonly 19 2 5 Specifying MTZ Columns and Map Type Because MTZ columns can be arbitrarily labeled AFITT s automation system requires that column labels be specified on the command line The following table gives the command line switches a description of the column and some usual column names found in MTZ files A map type must be specified for constructing the density with which to fit For example to load an MTZ file with Observed amplitude in column F Calculated Amplitide in column FC and Calcualted Phase in column PHIC using maptype 3Fo2Fc bin afitt e automate dir DATA grid mtz ligand lig pdb gz protein nolig pdb gz exit boxes lig pdb gz boxpadding boxonly FO F FC FC PHIC PHIC maptype 3Fo 2Fc 19 2 Advanced Automation 139 Table 19 1 MTZ Column Switches Switch Column Example Column Labels FO Observed Amplitude F FO FP FObs PHI Phase PHI PHIB FC Calculated Amplitidue FC FB PHIC Calculated Phase PHIC SIGMA Standard Deviation of Observed Amplitude FOM Figure of Merit FOM RFREE RFree FWT Amplitudes for weighted 2Fo Fc map PHIWT Phases for weighted 2Fo Fc map
21. 20 1 Afitt Mouse Map and Keyboard Shortcuts Function Mouse Buttons or Short Cut Zoom Wheel Y motion while holding Middle Button Y motion while holding Left Right Buttons Rotate XY Motion while holding Left Button Translate in XY plane Translate in Z plane Select Select Rectangle Select Add Select Grow Select Add Grow Select Rectangle and Add Change clipping plane Change far clipping plane Change near clipping plane Menu Adjust contour level Show Labels XY Motion while holding Left Button and Shift Wheel and Alt Click Left Button XY Motion while holding Right Button Click Left Button while holding Shift Click Left Button while holding Control Double Click Left Button Double Click Left Button while holding Shift Double Click Left Button while holding Control XY Motion while holding Right Button and Shift Wheel and Alt Control Shift Wheel and Alt Control Wheel and Alt Shift Click Right Button Wheel and Shift XY Motion and Control 141 21 1 CHAPTER TWENTYONE Release Notes Version 1 3 1 January 2008 Minor Bug Fixes 1 2 Coot mouse mode is more complete Popup right click menus now include the option to center in the 3D window and Spreadsheet this was in the Afitt 1 2 series CDX Chemdraw reader properly assigns hydrogens and generates 2D coordinates when given a 2D sketch This means that volume calculations are now correct for Chemdraw sketch files Molecules with only 2D
22. 5 Bookmarks The 3D display is capable of capturing and storing named bookmarks of the current display scene to enable viewing again at another time without having to set the scene up again by hand Bookmarks are saved in state files and are particularly useful when trying to communicate multiple pieces of information to other users All bookmarks have names and are stored in order Bookmarks can be created from the top level Book mark menu by selecting the Add option The order of the bookmarks can be organized by selecting the Organize option in the same menu The Bookmark menu also contains an Animated option which controls whether AFITT animates the transition between the current scene and the newly selected book mark A list of all the currently available bookmarks populate the rest of the menu Bookmarks can also be created by clicking on the Add Bookmark button in the 3D display Style toolbar see Section 5 7 1 The 3D display also provides a bookmark display widget see Section 5 8 2 which displays a clickable list of all the available bookmarks directly in the 3D display 5 6 Tiled Display The default behavior of the 3D display is to display everything that is Visible see Section 3 3 2 in the same coordinate space however there are often occasions when it is desirable to view everything in their own space This behavior can be obtained in Tiled mode also called Matrix mode or Multi Pane mod
23. 66 Chapter 5 3D Display e Symmetry operators This submenu lists the symmetry operators for the focused molecule It is possible to toggle individual symmetry operators on and off This option is very useful for e g producing the biologically active dimer using crystal symmetry If there are no operators listed in this submenu then either a no object has the focus b the focused object is not a molecule or c the focused molecule does not have symmetry information Currently symmetry information is only read from the CRYST1 record of PDB files e No symmetry color No special coloring will be used for symmetry replicates e Single symmetry color A single distinct color will be used for all symmetry replicates e Color by operator Each symmetry operator is assigned a color All symmetry replicates resulting from the same operator e g X Y Z have the same color no matter which unit cell they are in e Unique color Each replicate is given a distinct color so that no two replicates have the same color Since the number of replicates can change depending on the symmetry radius and or the current center this has the somewhat unfortunate side effect that occasionally translations cause every molecule to be recolored Notes after realizing symmetry if Show symmetry is left on there could be a very large number of molecules on screen as each new molecule is also symmetry expanded CHAPTER SIX Modeling Basics AFITT contains
24. 8 Display Widgets The 3D display provides a number of additional widgets which are drawn directly in 3D display as opposed to on the side or floating above it These widgets include an annotation widget a bookmark widget a data display widget and a depiction widget 5 8 1 Annotation It is often desirable when viewing molecules to make notes or simple annotations about specific molecules This can be done directly within the 3D display where the annotation widget appears like a transparent Post It note see Figure 5 9 The color of the widget as well as the font used can be specified in the application preferences Figure 5 9 Molecule with annotation The widget can be moved by clicking on the title bar and dragging it to the desired location The widget can be resized by grabbing the small triangular tab in the lower right hand corner of the widget and dragging until the desired size is achieved Clicking in the annotation widget will activate it for editing Editing can be accomplished by directly typing once the widget has been clicked or by clicking on the Edit button in the title bar Clicking on 48 Chapter 5 3D Display the Edit button will launch a more fully featured text editor which supports copy and paste operations see Figure 5 10 1 1 cox2 This is a COX 2 inhibitor and is potentially of interest What do you think Figure 5 10 Editing the annotation Annotations are also stored in the s
25. AFITT will identify many candidate locations for placing the ligand that will need to be analyzed e The reflection data should be refined without a previous ligand conformation While in many cases the same result will be generated if the ligand is fit to data refined with a previous ligand conformation AFITT will be biased towards the pre existing ligand conformation e Sometimes AFITT gets cluttered with too many results and becomes difficult to navigate Use the Edit Hide All menu option to clear up the data 6 5 3 List Window vs Spreadsheet Many of the same operations can be performed in the spreadsheet and the list window The spreadsheet has the ability to seperate different object types such as molecules proteins maps and blobs into different groups AFITT uses this cabability to help arrange the results of fitting ligands for instance and allows for sorting of results This is most often used when multiple stereo variants have been produced and the best fitting variant needs to be identified The list window contains every object loaded into AFITT and is a more general purpose browsing tool AFITT tries to generate a new list everytime a ligand fitting operation is initiated All relevant data for the fitting operation will be placed in the newly created list CHAPTER SEVEN Ligand Modeling The initial workflow displayed when opening AFITT for the first time is the Ligand modeling task The basic workflow for ligand fit
26. ALA 0 62 1 80 0 50 ARG 2 53 4 50 1 81 ASN 0 78 3 50 0 85 ASP 0 90 3 50 3 64 CYS 0 29 2 50 0 02 GLN 0 85 3 50 0 77 GLU 0 74 3 50 3 63 GLY 0 48 0 40 1 15 HIS 0 40 3 20 2 33 ILE 1 38 4 50 1 12 LEU 1 06 3 80 1 25 LYS 1 50 3 90 2 80 MET 0 64 1 90 0 67 PHE 1 19 2 80 1 71 PRO 0 12 1 60 0 14 SER 0 18 0 80 0 46 THR 0 05 0 70 0 25 TRP 0 81 0 90 2 09 TYR 0 26 1 30 0 71 VAL 1 08 4 20 0 46 ASX 0 84 3 50 2 25 GLX 0 80 3 50 2 20 Other 0 00 0 49 0 52 Surface Potential The surface potential scheme colors each vertex according to the potential value at that vertex using a red to blue gradient from the surface potential minimum to the surface potential maximum This scheme is different than the electrostatic scheme in that the coloring is based on the stored potential values as opposed to calculated potential values Typically surfaces generated within AFITT will not have any stored potential values However surfaces created externally using OpenEye s Spicoli toolkit for example can store values in the potential field and as such can be colored accordingly 5 11 3 Selection Vertices can be selected simplying by clicking on the desired location A line of vertices can be selected by selecting one vertex and then by holding down the Shift key when selecting another This starting selection can be grown and shrunk using the Selection pane in the Style Control see Section 5 11 3 Double clicking on the surf
27. Grids Surfaces 3D Display Ligand and Modeling Clicking on any of these 128 129 categories will update the right hand side of the widget to display the options corresponding to the selected category There are four buttons at the bottom of the dialog Clicking on the Restore button will restore the current preferences to the default ones Clicking on the Apply button will apply the current preferences to this run only and will not be automatically saved when AFITT is exited Clicking on the Save button will save the current preferences for this run and for future runs of AFITT Clicking on the Cancel button will close this dialog and will not apply any of the changed preferences Preferences are stored in a binary file preferences oeb in a user specific local directory on the computer currently running the application On Microsoft Windows 2000 and XP the preferences file can be found in C Documents and Settings USERNAME Application Data OpenEye AFITT 1 3 On Microsoft Windows Vista the preferences can be found in C Documents and Settings USERNAME AppData Local OpenEye AFITT 1 3 On all other platforms the preferences can be found in USERNAME OpenEye AFITT 1 3 While the preference file shares the same file extension as the OpenEye s binary database file it cannot be read into AFITT using the Open menu item in the File menu The preferences file is loaded auto matically when the application
28. Process Manager Processes Status Process Status Started Ended Hit structures will be in rocs_hits_l oeb Rocs Done 09 19 21 09 19 25 Query ref molecule written to rocs_hits_l oeb processed 467 overlays in 1 seconds 467 00 overlays sec 467 hits found Writing results Clear __Clear all Figure 13 2 Output generated by completed ROCS process 13 1 Processes When creating a new process or editing a current one there are a number of available options At the top of the editor there is a field for the process name which will be displayed in the list of available processes seen on the left as described above as well as a field for the actual location of the application to be run The Browse button to the right of this field can be used to find the location of the desired application Beneath this is a large text entry box which allows for specification of any desired command line argu ments to the application It is not necessary to enter the application name here For most applications at least one filename will have to be specified as a command line argument This can be done multiple ways If the file to be specified is always going to be located in the same location the filename can be entered directly into the text entry box as a command line argument If the location of the file is not likely to be constant a prompt can be specified as opposed to an actual filename To add a prompt click on the Ad
29. Supplied along with AFITT is a supplementary file named refmac_params py that contains the original values of these variables that can be used as templates for changing the parameters 9 2 Altering Refmacs Parameters 91 The following is the normal mode of running refmac3 When working with python there are two things to remember 1 the triple quotes are how python designates a block of text everything between two sets of triple quotes is captured in the variable include line breaks 2 the funny looking S F s indicates that a named variable is passed from AFITT to the refmac5 parameters These variables are e F The FC column from the original reflection file e SigF The Sigma F column from the original reflection file e RFree the RFree column from the original reflection file e weight The XRay weight to use e cycles The number of refinement cycles to used REFMAC_PARAMS make check NONE make hydrogen NO hout NO peptide NO cispeptide YES ssbridge YES symmetry YES sugar YES connectivity NO link NO refi type REST resi MLKF meth CGMAT bref ISOT ncyc cycles s scal type SIMP ESSE ANISO solvent YES weight IATRIX weight s monitor MEDIum labin F s SigF s RFree s labout FC FC FWT FWT PHIC PHIC PHWT PHWT DELFWT DELFWT PHDELWT PHDELWT FOM FOM RSIZE 80 END nu 92 Chapter 9 Protein Refinement
30. The following are the rigid refinement parameters make hydrogen NO hout NO peptide NO cispeptide YES ssbridge YES symmetry YES sugar YES connectivity NO link NO refi type RIGID resi MLKF meth CGMAT o rigid ncycle cycles s scal type SIMP reso 2 000 100 0 LSSC ANISO solvent YES weight MATRIX weight s monitor EDIUM torsion 10 0 distance 10 0 angle 10 0 plane 10 0 chiral 10 0 bfactor 10 0 bsphere 10 0 rbond 10 0 HESE 10 0 labin F s SigF s RFree s REFMAC_RIGID_PARAMS make check NON DJ labout RSIZE 80 END FC FC FWT FWT PHIC PHIC PHWT PHWT D ELFWT DELFWT PHDELWT PHDELWT FOM FOM CHAPTER TEN 2D Display 2D molecular depictions are provided using OpenEye s Ogham toolkit A large number of options are available in the applications preferences to customize how the depictions are drawn Depictions are drawn as black on a white background by default as seen in Figure 10 1 but other color schemes are available and can seen in Figure 10 2 VIDA 3 0 0 for OpenEye Worldwide File Edit View Data Style Bookmarks Tools Window Help vstle_ mateo a Is A gt amp O 3K XK List Window E 1 3 cox2 1 4 cox2 Name 2 cox2_good sdf gz E 1 1 cox2 1 3 cox2 fH 1 4 cox2 fH 1 5 cox2 fH 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 10 cox2 fH 1 16 cox2 1 17 cox2
31. a mech anism to allow the user to override the handling of certain formats SMILES PDB Mol2 XYZ and MacroModel The ability to change the flavor of a specific format can be done in the application preferences as seen in Figure 4 1 In addition to changing the flavor of a format there are a few other advanced options available when reading molecules including aromaticity model specification and conformer joining The desired aro maticity model to be applied can be specified in the pulldown menu next to the Aromaticity Model 4 1 Opening Files 25 label The Join Conformers checkbox controls whether or not adjacent molecules in an input file will be tested on reading to determine whether they are unique compounds or simply different confomers of the same molecule The specific test to be performed can be specified in the pulldown menu next to the checkbox For more specific details on the available aromaticity models and conformer tests please see the OEChem documentation It is important to note that because these advanced options are available through the application prefer ences they will be remembered by the application and automatically applied in future Open operations unless they are subsequently restored 4 1 2 Grids Multiple grid and map formats are supported for reading and include e OpenEye grids ASCII grids GRASP Delphi grids CCP4 maps XPLOR maps 4 1 3 MIZ Files AFITT can open s
32. a residue into alternate conformations is currently best accomplished by copying the residue in question and then pasting with this option 6 3 2 Adding and removing Hydrogens AFITT includes a Hydrogens menu to the edit menu which provides the capability of adding explicit hydrogens to molecules or converting explicit hydrogens to implicit hydrogens Both of these menu options will prompt for a molecule to operate on 6 3 3 Changing Residue Names AFITT allows for the changing of residue names for the Visible Selected or Marked residues This should be used with caution as changing residue names can heavily influence the running of external programs such as Refmac and CNS Xplor This option is mainly used to allow the changing of ligand residue names when there are two ligands with the same residue name but different structures If the two ligands have different structures but have the same residue name refinement dictionaries will most likely not be usable To change the residue name for the visible molecule select Edit Change Residue Name Visible At this point you will be prompted with a dialog to enter the new residue name Note This operation is not undoable 6 4 Popup menus AFITT includes a Center on selected option to the popup menu in the 3D viewer This option as the name suggests centers the view on the selected atoms AFITT also includes options to the context sensitive popup menu in the list widget R
33. basic operations for fitting small molecules to density To fit a small molecule to density you will need density a small molecule or connection table and optionally a protein The protein is used to mask the density where the ligand should not be placed Using the protein is highly recommended as this greatly facilitates the automated blob finding Each modeled unit density ligand protein has a set of controls that can focus the object or toggle its visibility Clicking on the name of the object will focus it clicking on the small box to the right of the object will toggle visiblility Clicking on the arrow will remove the item from the current model and 6 clicking on the button with three dots will browse the current repository for a new object Once a model has been loaded clicking on the Find Blobs button will attempt to locate regions of density suitable for the ligand The Find Blobs algorithm uses the current sigma value for the density If the Auto checkbox is selected then the searching algorithm will examine all density sigmas in an attempt to find a blob of the appropriate size It is recommended to use the Auto option as this has been tuned to searching density for standard ligand sized molecules The Auto checkbox is enabled by default however AFITT will remember the current state of the checkbox from session to session By default AFITT automatically selects blobs that are within 4A to the protein This distan
34. binary format v1 and v2 bin and oeb e MDL RDF and SDF 23 24 Chapter 4 File I O Preferences General Molecules Grids Surfaces 3D Display Custom Views x _ General Molecule Input Output Generic Input Flavors Aromaticity Model Join Conformers T Rings PDB Flavors PDB Flavors TER END ENDM Data Charge Radius Formal Charge Implicit Hydrogens Bond Order Rings Connect e Tripos MOL2 MOL2H Generic Output Flavors Aromaticity Model None Split Conformers Rings PDB Flavors v PDB Flavors Bonds Orders Both Charge O Radius Element Formal Charge TER HET Bonds Current Residues No Residues v OE Residues Y Order Atoms Restore lI Apply JM Save Jil Cancel Figure 4 1 Advanced molecule input and output options e Daylight SMILES canonical SMILES and isomeric SMILES e ChemDraw CDX e ISIS Sketch e MacroModel e MOPAC e PDB e XYZ As is often the case with file formats the meaning and use of certain fields within a format may change over time which can potentially lead to problems interpreting those files VIDA using OpenEye s OEChem toolkit makes its best effort to interpret all files correctly however it does provide
35. column contains the unique identifier assigned to that object by VIDA e VIDA Name This column contains the title of the molecule as sepcified in the input file e Visible This column contains an indicator green dot showing whether the molecule is Visible see Section 3 3 2 e Marked This column contains an indicator red flag showing whether the molecule is Marked see Section 3 3 4 e Locked This column contains an indicator padlock showing whether the molecule is Locked see Section 3 3 3 95 96 Chapter 11 Spreadsheet VIDA 3 0 0 for OpenEye Worldwide File Edit View Data Style Bookmarks Tools Window Help Focused vsble mrd al m CS Gi Ge a XK x ax List Window Depiction 0281 VIDA Name VIDA ID Family 1C50_uM Name og 1D S S cox2_good sdf gz 1 1 cox2 1 3 cox2 EA EE 1 16 cox2 1 5 cox2 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 10 cox2 1 16 cox2 1 17 cox2 ae gt gt AAT cox2 1 18 cox2 1 19 cox2 1 20 cox2 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 moet MP 1 18 cox2 1 29 cox2 1 30 cox2 1 31 cox2 1 32 cox2 1 33 cox2 1 34 cox2 1 35 cox2 Tae TR 119 cox2 1 37 cox2 14 20 cox All except List 3D Viewer 1 20 cox2 1 21 cox2 lt Molecules Proteins Atoms O cleee ccl eZecen2c3ccce c3 S Figure 11 1 Spreadsheet window 11 2 Sorting 97 Clicking on any dat
36. coordinates are not shown in the 3D window anymore Clearing all data repeatedly from the File menu could result in the result spreadsheet columns being removed Major Bug Fixes Fixed crash bug when finding blobs with no protein Fixed residue perception when forming covalent bonds Stereo enumeration now works from non connection table molecules Before it always used the input stereochemistry Improvements 1 142 Refinement dictionaries now update PDB atom names for molecules that never had them i e smiles strings Merging molecules now updates PDB atom names for molecules that never had them and attempts to add ligands to new chains residue numbers are incremented as a last resort 21 2 Version 1 3 0 143 10 11 12 Stereo enumeration now uses far less memory Blob finding now can find density at multiple contour levels Distance to protein slider added to ligand modeling widget This filters out blobs that are too far from the given protein Distance to protein flag added to automation system Molecule splitter dialog now automatically sets up the first split seperating the protein from the ligands Maps generated from MTZ files are now of higher quality Added Edit Menu to change residue names Ligands are added to new chain ids when possible 3D Toolbar indicators behave more sanely on Linux Residue names can now be changed Warning these names should not
37. data that are sampled at regular intervals Grids may contain electrostatic samples of spatial regions density samples and other potential fields A grid can have a variable number of contours Each contour has an index starting at 1 for the first contour Additionally every contour has its own individual color and a threshold value which can be positive or negative also known as the contour level There are three different display styles for grids contours Solid Mesh and Cloud 5 10 1 Grid Types As mentioned in the previous section there are multiple different predefined grid types that are supported including Electrostatic ET FRED Generic Difference Map and Regular Map Electrostatic 58 Chapter 5 3D Display Electrostatic grids are the only type of grid that can be actually be generated within AFITT as opposed to simply being visualized Electrostatic grids are created for individual molecules using OpenEye s Zap toolkit The creation of the grid requires the presence of partial charges on the input molecule If partial charges were already present on the molecule those will be used in the calculation However if no partial charges were present or the user decides to ignore them which can be done in the application preferences temporary partial charges will be calculated using MMFF94 or AM1 BCC 19 20 Ifa molecule has greater than 50 atoms MMFF94 charging will be done even if AM1 BCC was selected However fo
38. display and a Mouse toolbar at the left hand edge Other toolbars exist in the bottom and right areas 3D Bottom and 3D Right respectively but are empty and hidden by default They can be shown and populated via the use of scripting commands see Chapter 14 5 7 1 Style Toolbar The Style toolbar contains a single row of buttons which are primarily used to toggle the visibility of the 3D display widgets see Section 5 8 as well as the torn off Style Control panes see section 5 1 2 However the second button with the flagged folder icon creates a new named display bookmark see Section 5 5 based on the current scene and the last button toggles the use of stereoscopic visualization see Section 3 3 The buttons in the middle are organized into two groups The first group contains four buttons which toggle the display of the annotation widget the 2D depiction widget the data display widget and the bookmark widget respectively The second group contains five buttons which toggle the display of the torn off Style Control panes color selection style contours and graphics respectively This toolbar can be referenced in scripting commands using the name Style 5 7 2 Mouse Toolbar The Mouse toolbar contains a single row of buttons which control the behavior of the mouse in the 3D display Ignoring the sticky button the first button puts the mouse into the default mode which obeys all of the expected interac
39. for more rows to be displayed at the same time Certain molecular properties such as molecular weight can be added as new columns by selecting the desired option from the Molecular Properties submenu in the right click popup menu Some of these properties such as Energy might not be set on the molecule and as such the associated cell will contain an empty value See Section 11 5 2 for a complete listing of the molecule properties available for calculation 11 4 Organizing Columns Columns can be organized in the spreadsheet by selecting the Organize Columns option in the top level Data menu This will launch a dialog which can be seen in figure 11 3 Column Controller Order Depiction Visible Locked Marked VIDA Name VIDA ID Name Family 1C50_uM set Figure 11 3 Spreadsheet organization dialog The order of the columns can be controlled by selecting individual columns and then clicking on the up or down arrow buttons to change that columns position in the order Furthermore columns can be deleted by clicking on the button with the red X icon Please be aware that deleting a column will delete the associated data from all currently loaded molecules and spreadsheets 11 5 Creating New Columns 99 11 5 Creating New Columns Create Column PR Create Column Create Column New Table Column 5 Expression Math Functions mw cost EOLC VIDA 1D Evaluated on row 1 of Molecules Eval
40. fourth atom will create a permanent monitor and clear the anchor Monitors can be removed by performing a right click operation in the 3D display and selecting the Delete visible monitors option Monitors are also displayed in the List Window and can be deleted individually there see Chapter 12 Examples of the three types of monitors can be seen in Figure 5 9 4 5 9 5 Hydrogen Bonds Both internal intramolecular and external intermolecular hydrogen bonds can be displayed Hydrogen bonds are determined by typing all of the atoms as either acceptors donors both or neither Then the hydrogen bond energy is calculated between all acceptors and donors based on the ChemScore 18 5 10 Grid Visualization 57 scoring function which takes into account both interatomic distance as well as the coordination geometry This function provides a relative score between O and 1 0 for the energy of the hydrogen bond For all hydrogen bonds with an energy greater than 0 the bond is drawn as a dotted green line The thickness of the line as well as the spacing between the dots correlates to the bond energy Therefore thicker more solid lines have higher energy than thinner more dotted lines An example of both internal and external hydrogen bonds can be seen in Figure 5 17 a External Hydrogen Bonds b Internal Hydrogen Bonds Figure 5 17 Hydrogen bonds in a protein active site 5 10 Grid Visualization Grids are spatial arrays of
41. from the toolbar 3 6 Undo Redo 11 Fullscreen In addition to the standard layout described above the main window can optionally be displayed in fullscreen mode by selecting the Fullscreen option in the top level View menu Pressing the Esc key will exit fullscreen mode please note that clicking the mouse on the screen may be required before hitting the Esc key in order to ensure that the key press is registered When in fullscreen mode the main menu bar and status bar are hidden by default However they can be toggled on or off by hitting the F1 and F2 keys respectively The 3D display normally contains popup toolbars around the edge of the window These toolbars remain when in fullscreen mode to allow for easy access to display functionality For more details on these toolbars see Chapter 5 3 5 2 Peripheral Windows In addition to the main window there can be multiple peripheral windows docked around the edges of the main window or as separate floating top level windows Furthermore multiple windows can be placed on top of each other in a tabbed region Currently multiple peripheral windows including the potential main windows are available e 2D Display see Chapter 10 e 2D Preview see Chapter 10 e 3D Viewer see Chapter 5 e List Window see Chapter 12 e Process Manager see Chapter 13 Scripting Window see Chapter 14 Spreadsheet see Chapter 11 Style Control she Chapte
42. is specified to handle the output file the actual filename can be obtained by using the keyword FILE in the function specification This keyword will be substituted with the real filename at process completion time before the Python function is called Finally there is a checkbox at the bottom to specify whether or not this is a default output The default output entry is used when constructing pipes and will be discussed in Section 13 2 13 1 3 Adding Prompts As described in Section 13 1 there are two types of prompts that are available for specification on the command line a text prompt and a file prompt The Text Prompt Editor see Figure 13 5 a allows the user to add a text prompt to the process as well as to specify what the caption of the prompt will be Specification of an informative caption is helpful in allowing the user to know what information is being requested E Prompt Text PR E Prompt File Please specify desired caption Please specify desired caption l i OK Cancel a Text Prompt Editor b File Prompt Editor Figure 13 5 Prompt Editors 13 2 Pipes 117 The File Prompt Editor see Figure 13 5 b allows the user to add a file prompt to the process as well as to specify what the caption of the resulting prompt will be Specification of an informative caption is helpful in allowing the user to know what specific file or type of is being requested The result of both of these prompts are pass
43. it is visualized in AFITT including the default number of contours and their levels and the display style as well as the color of those contours For more details on the individual grid types see Section 5 10 1 In the bottom area there are three individual rows of buttons In the first row the first three buttons turn on the display of electrostatic grids molecular surfaces and accessible surfaces respectively These specific grids and surfaces are considered display properties of their associated molecules and therefore are not displayed in the List Window see Chapter 12 Independent non property versions of these can be created from the relevant options in a right click menu see Section 5 1 1 The next two buttons turn on the display of protein ribbons and c alpha traces respectively for more information about these protein specific displays see Section 5 9 3 The next button turns on the display of hydrogen bonds by making all of the molecules in the current scope to be hydrogen bond targets Being a hydrogen bond target means that when it is visible it displays any hydrogen bonds made between it and any of the other molecules that are also visible The last button in the row toggles whether or not internal intramolecular hydrogen bonds are shown In the second row the first three buttons turn off the display of electrostatic grids molecular surfaces and accessible surfaces respectively The next two buttons turn off the display o
44. modeling specific operations in the various menus as opposed to generic visualization operations described in Chapter 5 these are described in detail here and in the following chapters 6 1 Tasks To facilitate ligand and protein fitting workflows AFITT divides workflows into Tasks Each Task has a basic set of windows related to the current task For example the ligand fitting workflow contains the Ligand Modeling window the List Window the 3D Window and the Spreadsheet These workflows can be selected from the Task menu or from the Layout pulldown It is highly recommended to use the supplied task workflows although they can be modified at will 6 1 1 Task Menu The task menu changes the current workflow The available workflows are 1 Ligand This workflow is optimized for fitting ligands to crystallographic density 2 Protein This workflow is optimized to fitting protein residues to density 3 Refinement This workflow helps to view the results of launching external refinement packages such as refmacS 6 2 File menu Two modeling specific menus are Add from SMILES string and Write refinement dictionary 67 68 Chapter 6 Modeling Basics The Write refinement dictionary menu has options to write either a refmac or CNS X PLOR dictionary entry for the currently selected molecule s The user will be prompted for the path and filename prefix which will then be used to produce cif and pdf files ora
45. of the primary navigation and organization mechanisms in AFITT It displays all of the currently loaded files and their contents molecules grids surfaces etc into hierarchical lists for easy browsing and organization The List Window can be seen in Figure 12 1 and contains multiple columns for controlling the various states and properties of the associated objects The first column displays the name The second column with the green sphere icon at the top is the Visible state column see Section 3 3 2 The third column with the padlock icon at the top is the Locked state column see Section 3 3 3 The fourth column with the flag icon at the top is the Marked state column see Section 3 3 4 The fifth and last column contains the unique identifier assigned to that object 12 2 Browsing Every list in the List Window as well as many of the other objects can be expanded in the List Window to reveal further detail or the members contained within them A small box with a sign inside to the left of an object s name indicates that that object can be expanded Molecules for example may be expanded to reveal conformers molecular structure or potentially other related children objects such as surfaces or grids A single mouse click on the box will expand that object to reveal its contents Double clicking on the object name will turn on in place renaming of that object 107 108 Chapter 12 List Management List Window Name
46. or by varying torsion dihedral angles of shape matched ligand conformations 8 are unable to prevent creation of high energy sometimes even chemically unrealistic ligand models Many such density fitting artifacts have been analyzed resulting in about 10 of ligands deposited up through 2004 including high amounts gt 10kcal of strain 9 AFITT uses a combination of OpenEye s shape fitting technology and a force field to maintain proper chemistry while fitting the ligand to density 10 AFITT also automatically enumerates stereo chemistry and finds the best stereo variant that fits the supplied data These techniques have been extended to include residue and side chain fitting facilitating protein model ing Additionally AFITT generates quality refinement dictionaries 11 including detecting and preparing covalent bonds for input to refmac5 12 or cns x plor 13 refinement programs In summary AFITT provides e Fast accurate ligand fitting to density e Protein residue and side chain fiting to density e Generation of high quality refinement dictionaries 6 Chapter 3 Getting Started 3 2 General Concepts There are a number of general concepts that are important to discuss early in order to explain how user interaction with AFITT works AFITT can real space fit both ligands and proteins using a variety of techniques such as best rotamer search rigid body and MMFF Shape fitting These operations can be performed at
47. point a file browser will appear for the location where the dictionary will be written For example to write a REFMAC refinement dictionary for all visible molecules choose File Write refinement dictionary gt REFMAC gt Visible This is the most common procedure for writing out refinement dictionaries Dictionary generation uses the MMFF94 forcefield to form geometric constraints for ligands and proteins under refinement When writing out REFMAC dictionaries the following residues will not be written since REFMACS already has geometric constraints this also means that any ligand that needs to be written out should not have one of the following residue names 78 Chapter 7 Ligand Modeling ALA TLE TRP MVA TYS SHP DIP GAL b D ARB CEG b D 1GN 5CM AFI APH ASX BOG C5X CHP CSS DAB DDB DIV DSE EJT ARG LEU TYR IVA CGU CSO BAL Ir GLC RIP HOH IMG SIT AGL APM B7G BRU C6C CLB CSW DAF DDL DLF DSN EMP Table 7 1 Residues Names Not Written to Dictionary ASN LYS VAL DFO STA ETA A C MAN GLC b D ABE 5GP 1PA SIU AGM ARA BCS BTA CAN CLD CSX DAG DFX DLY DSP EPU ASP MET ACE NME ILG TFA Ad Cr MAN b D XYL RAM AMP 2AS 5NC AHO ARM BDG BTC CAR CME CTH DAR DGL DMT DSR EYS CSH MSE FOR AHT OCS ANI C G NAG DRB MAL IMP 2GL 8BR AHB ASG BGL BTR CAS CPC CUC DAS DGN DNP DTY FAG CYS ORN ABA PTR KCX MPR Cd Gr NAG b D
48. rendered as reentrant can be distinguished from non reentrant grids by the absence of grid corners which define the extents of non reentrant grids 5 11 Surface Visualization Surfaces are infinitely thin three dimensional connected regions that represent objects such as molecular or accessible surfaces of molecules Surfaces can be selected or scribed cropped colored and made transparent in part or in whole 5 11 1 Display Styles Surfaces can be visualized in one of three display styles Solid Mesh and Points These individual style can be seen in Figure 5 19 60 Chapter 5 3D Display a Solid b Mesh c Points Figure 5 19 Surface rendering examples showing a solid rendering b mesh rendering and c point rendering 5 11 2 Color There are a number of surface specific coloring schemes in addition to the standard single color scheme The following schemes are discussed below atom color concavity curvature distance electrostatics grid hydrogen bond potential hydrophobicity and surface potential Atom Color The atom color scheme colors each vertex on the surface using the color of the nearest atom to that vertex in the molecule that the surface was created from see Figure 5 20 b This scheme will not work if the surface was not created from a molecule and also if that molecule is not currently present Concavity The concavity scheme colors each vertex on the surface according to how concave the su
49. the bottom input area It also displays any warnings or errors generated during the run of the application The input component is the single line text entry area at the bottom of the window On the input line the user can directly type in Python commands to be run by AFITT The scripting input line is intelligent Hitting the Tab key at the beginning of a line will indent the entry line to the previous line s identation if there was any indentation at all If there was no indentation the line will indent five spaces However hitting the Tab key after typing a portion of a command will cause the scripting line to expand out that command as far as possible without introducing ambiguity For example hitting the Tab key after typing Tool will expand out to Toolbar At this point hitting the 120 Chapter 14 Scripting Tab key again will result in a listing in the output display above of all the commands that begin with Toolbar see Figure 14 1 Finally hitting the Zab key when a command is fully expanded will display the API documentation associated with that command in the output display 14 5 OEChemLite oechemlite is a Python module that ships with AFITT which enables access to the currently loaded molecules via the scripting interface oechemlite is distinguished from OpenEye s PyOEChem mod ule in that it oechemlite provides only the PyOEChem functions that are not capable of making modifications to the molecules F
50. this example it never will so hit the stop bottom in the lower left of the screen to abort the script 19 1 2 Exiting AFITT When Finished To exit AFITT when finished processing all the current data simply add the exit flag to the command line bin afitt e automate grid DATA 1115 grd ligand DATA 1115 lig pdb gz protein DATA 1115 nolig pdb gz exit 19 1 3 Writing a Log File Since AFITT is mainly a GUI application the logs are not written to the terminal To keep a log file use the logfile option bin afitt e automate grid DATA 1115 grd ligand DATA 1115 lig pdb gz protein DATA 1115 nolig pdb gz xit logfile my log 19 2 Advanced Automation The automation system can scan multiple directories when looking for data to fit This is accomplised using the dir option 19 2 1 Using WildCards for Directories Because most shells have globbing that is they expand wildcards in filenames such as AFITT also uses the character to define wildcards on the command line For example using 19 2 Advanced Automation 137 dir DATA will tell afitt to search every directory in the subdirectory DATA The same wildcards can also be used to specify ligand protein and grid targets However the grid ligand and protein wildcards are only applied within a directory For example bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit will sea
51. to the top allowing easy identification of the correct stereochemistry 3 12 3 What To Do With Too Many Blobs Sometimes too many blobs will be found when searching density To filter out undesired blobs when searching select an atom or residue in the binding pocket and set a distance halo Any blobs that are found outside this distance will be rejected See 5 11 3 for more information on setting the distance halo 3 13 Protein Task 17 3 12 4 Working With Poor Density Occasionally the density is too poor to find a blob using the automated process In this case there are three options 1 Split the Protein Sometimes the protein has ligands embedded in the structure You can use the split options of the Edit menu to separate the ligand from the protein 2 Use a Box Under the task menu there is an option labeled Add Box This prompts for a molecule and then for an optional padding The extents of the molecule and the padding are used to generate a volume of space that constrains the search For example if the location of the active binding site is known the residues around the active site can be selected and used to define the box When a box is created the region is visualized in the 3D window At this point the box can be used as a fitting region or clicking on Find Blobs will only search the density inside the box Note multiple boxes can be used simulataneously 3 Picking Density Using the B
52. to go Like the undo history this menu only displays the 10 most recent undone operations but the redo history may be much greater and can be revisited to go further 3 7 3D Window Usage Most of the time spent in AFITT will be spent working in the 3D viewing window which is the central area of the application This window shows the currently visible objects such as density grids molecules proteins and blobs Left clicking on most items will select them For instance left clicking on a blob will select that blob and focus it The selected blob will then be used to extract the density region that will be used to fit the small molecule Mouse Map Mouse operations can be found in chapter 20 3 8 Spreadsheet Usage The spreadsheet shows more details about the objects that are loaded in AFITT It is seperated into five tabs Molecules Proteins Maps Blobs and Results The Molecules tab displays all of the small molecules that are currently loaded in AFITT The Proteins tab displays all of the proteins currently loaded in AFITT The Maps tab contains the currently loaded densities and grids The Blobs tab shows the auto detected blobs and surfaces Finally the Results tab contains the small molecules that have been fit to density For more information about using the spreadsheet see Chapter 11 3 9 2D Preview Usage The 2D Preview window shows either a 2D depiction of the small molecule or the sequence of the protein depending on which one
53. to the interactive symmetry display information entered here will also be used for the CRYST1 record if the molecule is written as a PDB file e Show unit cell This toggles the display of the crystallographic unit cell of molecules which have symmetry information specified e Show symmetry This toggles the display of symmetry related molecules hereafter called repli cates If enabled all visible molecules with symmetry information will display symmetry e Realize symmetry The symmetry display above is only for display purposes If the user wishes to actually expand a molecule to produce one with real coordinates for the replicates the symmetry must be realized This option will prompt the user to determine if the current molecule s should be symmetry expanded in place or if a new copy of the molecule s should be created and then symmetry expanded Only the symmetry operators currently enabled are used in the expansion e Symmetry radius All symmetry operators which would produce a molecule within the symmetry radius of the screen center are shown With a large symmetry radius this may result in more replicates than symmetry operators as each operator may be used more than once with different translations As a special case if the symmetry radius is 0 0 then each operator is used exactly once with a translation which places the center of mass of the molecule in the default unit cell i e the cell at the origin
54. 1 73 73 73 73 74 74 76 80 80 81 81 82 84 85 86 87 87 88 89 89 90 10 11 12 13 14 15 16 17 18 2D Display WO Interaction e cecep Be ab ee hw he Hw a a Ge Ow a wher ww We Sow Se eS Spreadsheet Pid Basic Usage ceci ai u kied wauni ae Aa wide at di ded amp Bk amp Gok eae dd TLZ Sorin o s dec eh aay we rh we oe a ee a wa te es ae ew eh we Be we He Rt ia 113 Displays Dala o ads a er A ede Sow dom Are cada as 114 Organize Columns teca aneia aa a deka Bele bere e be dew Sead ag ahaa al at 11 5 Creating New Columns cores eee ek a Ee ee a ee es PLG Filtering s sec de De SO ee ERE REESE Ae we We Stanis escasa aa Oe se SERS PE wa SARS ES 11 8 Formatting Columns lt lt sis 6a a aoe Oa ke ee eG ee EERE Ea RE wo 119 Importing EXPORAS p e a aa a be ee a Ba oe owe a ee A G List Management 12M TIniroducion aria earr ew Be Ree ee a we Ee a amp ee T 12 2 Browsing steed oa Sate PRR be ee EE ERE ERS EE ee be es A 2c oe NE 12 4 List COMMMS haces aida aA eh nck WR aA a cl GR A am S 12 3 List Manipulation s yo o g Gee hee ok we ee oe Pee ae ER ae eG 120 Let Wombat oe ete ee peel eee RS we OS Process Management WSs NE 1322 PIPES a hd a ae eee Ree A ee Ge AS ee a Scripting Tcl Joumal Files aiis asa a Soy Be A eee as ed we ee ia 14 2 Startup Pile ca eee a be LR Oe ee eee Bae EE Se ee a es 14 3 Scripting Manual s s s com as ee ba oe ER ER Eee ee we es 144 Seripti
55. 1 18 cox2 1 19 cox2 1 20 cox2 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 1 29 cox2 1 30 cox2 1 31 cox2 1 32 cox2 1 33 cox2 1 34 cox2 1 35 cox2 1 36 cox2 1 37 cox2 1 38 cox2 1 39 cox2 1 40 cox2 1 41 cox2 1 42 cox2 iH 1 43 cox2 2 20 cox2 3 5 cox2 eee Celeee nic2ccelcc2 Fic3cce cc3 5 O O C 0000000089 K amp Be El EE 00008 Figure 10 1 2D Depiction 93 94 Chapter 10 2D Display 1 1 cox2 F He d A a Color on Black b Color on White c White on Black Figure 10 2 Depiction color schemes As might be expected 2D depictions of this nature only make sense for small molecules lt 255 atoms in this case For proteins an amino acid sequence view is displayed instead see Figure 10 3 2D Viewer in H IVEGSDAE IT GMSPWOVMLFRSPOELLCGASLISDRWVLTAAHCLTENDLLVRIGKHSRIRYRNI 79 EKISMLEKIYIHP DRDIALMEBERENARSDVUIRENODE DRETLLOAGYEGRYTOWGNL 144 KRETWEGOPSYLOVVYNLP IVERPYCKDSTRIRITONMFCAYKKRGDACEGDSGGPFVMESNNRWYO cs ii E GCRDGKYGFYIHVFRLEEWIQKVIDOFG ain DFEEIPEEYLQ hain L CGLRPLFERKESLED Figure 10 3 Amino Acid Sequence 10 1 Interaction 2D selection behaves much like selection in the 3D display see Section 5 11 3 Left clicking will select an individual atom bond or residue if in sequence mode When viewing a depiction the right mouse button allows lasso style selection as in the 3D display When viewing a sequence hol
56. 1 6 Filter expressions are very similar to column generation expressions see section 11 5 except that the return value of a filter expression determines whether or not that row will be included in the new spread sheet Rows for which the expression returns either False or O will not be included 11 7 Statistics Statistics for each numeric column can be viewed in the spreadsheet window The display of statistics is controlled by toggling the Show Statistics option in the top level Data menu Toggling this option controls the display of statistics for just the currently viewed spreadsheet The following statistical information is computed for each numeric column 102 Chapter 11 Spreadsheet Create New Table New Table 1cso Expression Math Columns 1c50_um COL ICSO_uM lt 1 0 Evaluated on row 1 of Molecules False VIDA 3 0 0 for OpenEye Worldwide Figure 11 5 Filter dialog View Data Style Bookmarks Tools Window Help vsbe marked a m E Sar OKK Depiction es VIDA Name VIDA ID 150 MA gt 4 75 cox2 Molecules Proteins Atoms 1C50 COctee ce e1 F e2ne en2e3eee c3 5 OK O C C F FIFE Figure 11 6 A filtered spreadsheet 11 8 Formatting Columns 103 e min The minimum value of the column e mean The maximum value of the column e stddev The standard deviation of the column e sk
57. 2D display as well as the 3D display 3 3 3 Locked State The Locked state is essentially the same as the Visible state see Section 3 3 2 except in the situation where the application is in tiled mode in which case each Locked object will be displayed in every pane of the 3D display However a Locked object can also be simultaneously Visible and or Focused see section 3 3 1 in which case it will be displayed in its own individual pane as well as every other pane in the 3D display 3 3 4 Marked State The Marked state is simply an indicator of user interest in the associated object The Marked state can be used to help filter data as well as to specify the desired input to application functions For instance the Marked state can be used to specify which molecules out of a large list are saved Finally when certain operations generate too many results to be displayed at one time the Marked state can be used to indicate the results of those operations 3 35 Selected State The Selected state is a special property of the Visible state see Section 3 3 2 Only objects that are Visible Focused see Section 3 3 1 or alternatively Locked see section 3 3 3 can be Selected Much like the Marked state see Section 3 3 4 the Selected state is a indicator of interest and provides an input set to application functions Unlike the Marked state the Selected state is more transient and is easily cleared The act of selection is usually performed i
58. 2F GCN GLS GSR HMA IGL KAN MAI MDP MHO MUR NGL OMT PGP PPN RHA SCS SMC TBM TOA TYQ G4D GFP GLX GSS HMF IGU LLY MA2 MEN MHS MXY NGS ONL PGY PR3 RIA SEB SME TDG TOC TYV G4S GHP GLZ GT9 HPH TIL LNO MA3 MF3 MIA NSM NMC OPR PHA PRR RIN SEC SNC THC TPO TYY CHAPTER EIGHT Protein Modeling The Protein Task gives complete control over atom positions in density The initial view of the protein mode is seen in 8 1 While the initial view of Protein Mode is intimidating there are only a small handful of operations that are used regularly These operations also have hot keys and key stroke combinations to facilitate usage for advanced users Note For previous users of AFITT the Modeling Mode has been removed Modeling operations can be performed at any time and can be undone by using the Edit Undo menu or Ctrl Z keystroke As seen in 8 1 the Protein Mode task is divided into several user interface components e List Window controls states of molecules e 3D Window visualizes results of operations Style Window performs modeling and visualization operations e 2D Preview Allows easy access to residues e Ramachandran Plot Shows overall validation of protein These components are described in detail below 8 1 List Window On the left side the List Window shows what objects are currently loaded in AFITT and their visibility and marked states 80 8 2 3D Windo
59. 50 Chapter 5 3D Display b Stick d CPK e Stars Figure 5 11 Molecule rendering styles MM Atom Colors We ASK AK OF Om HO HEH NB Restore Ok Cancel Defaut Restore Ok Cancel a Atom Color Palette b Residue Color Palette H 7 i Figure 5 12 Dark background atom and residue color palettes The amino color scheme is a protein specific scheme which colors atoms according to their individual residues The residue colors are listed below 5 9 Molecular Visualization 51 ASP GLU Bright Red 230 10 10 CYS MET Yellow 230 230 0 LYS ARG Blue 20 90 255 SER THR Orange 250 150 0 PHE TYR Mid Blue 50 50 170 ASN GLN Cyan 0 220 200 GLY Light Grey 235 235 235 LEU VAL ILE Green 15 130 15 ALA Dark Grey 200 200 200 TRP Purple 180 90 180 HIS Pale Blue 130 130 210 PRO Flesh 220 150 130 Others Tan 190 160 110 BFactor The bfactor color scheme is a protein specific scheme which colors atoms according to the bfactor value using a fixed blue to red gradient between 0 and 100 Carbon The carbon color scheme colors all of the carbon atoms in the current scope the same color which is specified by the user Chain The chain color scheme is a macromolecule specific scheme which assigns a unique color to each of the macromolecular chains CPK CPKNew The CPK color scheme is an element specific scheme based on the colors used in the popula
60. 69 SER Medium Orange 255 112 66 VAL Light Purple 255 140 255 THR Dark Orange 184 76 0 LYS Royal Blue 71 71 184 ASP Dark Rose 160 0 66 ILE Dark Green 0 76 0 ASN Light Salmon 255 124 112 GLU Dark Brown 102 0 0 PRO Dark Grey 82 82 82 ARG Dark Blue 0 O 124 PHE Olive Grey 83 76 66 GLN Dark Salmon 255 76 76 TYR Medium Brown 140 112 76 HIS Medium Blue 112 112 255 CYS Medium Yellow 255 255 112 MET Light Brown 184 160 66 TRP Olive Brown 79 70 0 ASX GLX PCA HYP Medium Purple 255 0 255 A Light Blue 160 160 255 C Light Orange 255 140 75 G Medium Salmon 255 112 112 T Light Green 160 255 160 Backbone Light Grey 184 184 184 Special Dark Purple 94 0 94 Default Medium Purple 255 0 255 5 9 3 Proteins In addition to the standard display styles the two protein specific displays of C Alpha Traces and Ribbons are supported Examples of these display styles can be seen in Figure 5 13 Given that protein files often inevitably contain many extra components that are not necessarily of interest to the user waters for example the ability to hide the display of non bonded atoms is provided to ease the viewing of proteins without having to manually edit the input file An example of a protein displayed with and without waters can be seen in Figure 5 14 Another common scenario when working with proteins is that the input file contains a large multimer whi
61. AFITT User Manual version 1 3 OpeneEye Scientific Software Inc January 23 2008 9 Bisbee Ct Suite D Santa Fe NM 87508 www eyesopen com support eyesopen com Copyright 1997 2008 OpenEye Scientific Software Santa Fe New Mexico All rights reserved All rights reserved This material contains proprietary information of OpenEye Scientific Software Use of copyright notice is precautionary only and does not imply publication or disclosure The information supplied in this document is believed to be true but no liability is assumed for its use or the infringement of the rights of others resulting from its use Information in this document is subject to change without notice and does not represent a commitment on the part of OpenEye Scientific Software This package is sold licensed distributed subject to the condition that it shall not by way of trade or otherwise be lent re sold hired out or otherwise circulated without OpenEye Scientific Software s prior consent in any form of packaging or cover other than that in which it was produced No part of this manual or accompanying documentation may be reproduced stored in a retrieval system on optical or magnetic disk tape CD DVD or other medium or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise for any purpose other than for the purchaser s personal use without a legal agreement or other written permission granted by Ope
62. ASP and SPOCK The AFITT query language provides a powerful query syntax where atoms bonds conformers and molecules may be queried to see if they match certain properties Triangles vertices and surfaces may be similarly queried A fairly simple example is the following command Select Ch A amp amp rn 10 This command will select all atoms in chain A and in residue 10 for every molecule currently in memory A more complex query is the following Select id 5 amp amp r Shydrophobic rn 10 50 which selects every hydrophobic residue or residues with numbers between 10 and 50 but only matches atoms in the molecule with ID 5 As the examples indicate the general syntax of the query language based on expressions Each expres sion consists of a property a mathematical operator and a value Expressions may be combined using a syntax similar to the C and or Python programming languages Boolean operations are supported via the and operator amp amp as well as the or operator Nesting of parts of the query is possible via parentheses and The operators can also be spelled out as in Python and and or are valid for boolean operators 15 2 Operators AFITT s query language supports a full range of mathematical operators for testing a property s value against the value s specified in the query string Supported are gt lt gt and lt 122 15 3 Lists and Ranges 123 15 3 Lists and Ra
63. All scope can be halted if desired by clicking on the Stop button immediately to the left of the progress bar The current scope is displayed in and can be modified from the main application toolbar 3 4 1 Annoyances with Scope The largest problem that users have with scope is the difference between a focused object and a visible object A focused object is always visible but because another molecule may be focused next say by selecting an atom the previous focused object may disappear since it is no longer focused To prevent this simply make the molecule visible then focusing on another molecule will not make it disappear To make an object permanetly visible click on the dot next to the object in the List Window or the Ligand modeling window See the Layout section next for more details 3 5 Layout The layout of the application should be relatively familiar to most users AFITT provides a menu bar with many standard as well as specific menus a toolbar for common operations a central main window and a number of peripheral windows An example of the layout can be see in Figure 3 2 3 5 Layout 9 File Edt Task Data Style View Window Help visble mated at a ga Ce Style o Modeling TRTA Ha i PAEL ae ae a a O 1428 pdb ligand PR WWE OHMIC e 10 0 99 999 HEE Regular Map 2Fwt 1A28_prot mtz Difference Map None PLINLIMSTE PDVIYAGHINTK EDT SSSLLT SINOLGEROLLSVVEMISE SLPGE RNLH
64. By Not Marked option creates a list with all of the objects from the list which are not Marked By Query generates a dialog which prompts for a substructure query that 1f the objects match those objects will be placed in the new list The By Not Query behaves the same way except that it puts the objects that do match the query into the new list The last way to create a new list is from the Find menu entry in the Edit menu This mechanism searches all of the currently loaded molecules by either Amino Acid Sequence SMARTS a regular expression against the molecule title or by a Query molecule as a substructure search After clicking the Search button the matching results will be displayed on the right hand side of the dialog The 112 Chapter 12 List Management Sees O Figure 12 5 List navigation buttons entire set or just specific individual results can be selected within the dialog to populate the new list 12 5 2 Deleting from Lists As previously mentioned objects can also be removed from lists There are two ways to remove an object from a list The first mechanism is to delete the object by selecting the Delete option from the right click menu Deleting an object will remove it from ALL of the lists to which it belongs Choosing the Remove from List option from the same right click menu will remove the object from the specified list only leaving it perfectly intact in any other lists that
65. LTKLLONLHDLVKOLHLYCINTF IQSRALSVEF PEM LIPPLINLIMSTE PDVIYAGHINTKPD SSSLLTSINOLGERQLLSVVKWSKSLEGERNLHIDDQITLI OY SWMSLMVEGLGWRSY KHVSGOMLYFAPDLILNEQRMKESSE EVKLOVSOEEFLOMEVLLLINTI PLEGLRSOTOFEEMRS Sy RELIKALGLRORGVVS SORE YOLTRLLONLHDLVEQLHLYCINTELOSRALSVEF PEM 2D Preview Scripting Window CC O C H ICCLC SH E C CC C H a C HJ2cCc4 CC O CC C S4C IC Figure 3 3 Changing the layout by dragging File Edt Task Data Style view Window ma ye NZ Focused Visible Marked all MESS List Window ox Style Name ocr ir Modeling E o a o a a aoa rs y ae S ll dd A a E Bae os 1A28 pdb igand PR a DONdOUEl cocoa a e 10100 99 999 EJEJETElE q Regular Map 2Fwt 1A28_prot mtz lg Difference Map None l Transparency 5 X Ramachandran A Wichardkon General y QUT PPLINIIMSTEPDVIYAGHINTKPDTSSSTLTSINOLCEROLI SVVEWSKSLEGERNLHTDDOT TLTOY SUMSIMVECNRSYKHVEG MLYEAEDITINEORMKESSE QEEFLOMEVLLLINTI PLEGLRSQTOFEEMRSSYIRELIKALGLRORGVVSSSOREYOLT ILVKQLHLYCINTFLQSRALSVEF PEM EVELOVS PRILACMVKELLEHKE LIPPLINLIMSTEPOVIYAGHDNTKED SSSLLTSINOLGEROLLSVVEWSKSLPGERNLHIDDOLTLI GY SUMSIMVEGLGWRSYREVSGOMLYFAPDLTINEORMKESSE AR SLIM BORE VAL VS CRES LOMKVLLLINTI BLE GLRS ONE EEMRSSY TRELIRAL GLROMGV VESS0RE YQLTALLONLADLVAQLALYCINTE LOSRALSVEE PEM 20 Preview Scripting Window CC 0 C H ICC C SH 2 C CC C H gt C H 2ccC4 Cc O CC C s4C c Figure 3 4 Changing the layout
66. ONE RECEPTOR w Auto Find Y Show Modeled 11 000 risa o VIDA Name volume gayam oO Molecular Volume 174 269 pr e a EA y a a Y Model Ligand List Window Spreadsheet X 2DPreview ax 1A28 pdb lig e aif VIDA Name VIDA ID 1 0 1A28 pdb ligand 8 Molecules Proteins Atoms Maps Blobs Results Spreadsheet Scripting Window CC 0 C H 1CC C H 2 C 1 CC C HJa C HJ2CCC4 CC O NCCLC B4C C Figure 7 1 Ligand mode layout essary to supply ligand density small molecule If the density includes an already or partially modeled protein this greatly assits in locating the appropriate ligand density The protein is used to mask the density where the ligand should not be placed Using the protein is highly recommended as this greatly facilitates the automated blob finding Each modeled unit density ligand protein has a set of controls that can focus the object or toggle the visibility Clicking on the name of the object will focus it clicking on the small box to the right of the object will toggle visiblility Clicking on the arrow will remove the item from the current model and e 99 clicking on the button with three dots will browse the current repository for a new object Once a model has been loaded click on the Find Blobs button to attempt to find a region of density suitable for the ligand By default the Find Blobs algorithm uses the current
67. Once the desired file has been chosen another dialog will appear which allows the user to select which spreadsheet and which columns in that spreadsheet should be exported For more specific details on exporting spreadsheet data please see Section 11 9 4 5 2 Images Screenshot A screenshot of the current main window see Section 3 5 can be exported in an variety of image formats to a user specified file by selecting the Screenshot option in the Export submenu in the File menu This action can also be achieved by clicking on the button with the camera icon in the application toolbar Selecting this option will launch a dialog showing a preview of the screenshot to be taken as seen in Figure 4 6 The user can also adjust the desired image resolution as well as specify whether or not to capture the image in black and white which can be useful for publication purposes The output file is specified at the bottom of the dialog POV Ray The scene in the 3D display can be exported to a POV Ray input file which can be used to generate very high quality and resolution images To export a POV Ray file select the POV Ray option in the Export submenu in the File menu The application will then prompt the user to specify a file to which the scene will be saved 32 Chapter 4 File I O Screenshot Dimensions pixels 649 x ss3 Maintain original aspect C Black and White Filename Devel Trunk debug apps vida screenshot png
68. RIB LAT UFP 2MA A23 ALM ASK BGP BUC CAY CPR CYG DBY DGP DO2 FCA GLN PHE BOC PCA SAH DAM G U SIA FRC SUC UMP 3DR AAR ALN ASL BHD C3X CBI CSB CYM DCG DHI DOH EDC FCB GLU PRO BMT HYP SAM ACB Gd Ur FUC FRU GCU SGN 3FM ABH ALO ASM BLG C4X CCS CSD DIP DCM DHN DOM EEB FCY GLY SER SAR INI SEP ADD YG FUC a L XYS GCU b D 1AR 3GA AEI AMG ASO BMA CSC CEA CSE D3 DCY DHU DPP EFC FGL HIS THR MLE NLE LLP CXM Td PSU GAL XLS CEG 1GL 4SU AET AMU ASQ BNG CSP CH CSP D6G DDA DIL DRI EHP FLA 7 8 Writing Refinement Dictionaries 79 FME G6P GL3 GMA GUP HSO IMC LOL MA4 MFB MIS NAA NP3 OTG PHD PRS RON SEG SOC THO TRF UAP Table 7 2 Residues Names Not Written to Dictionary continued FMP G7M GLA GMH H2P HTR IML LTA MA6 MFU MLY NAM NPH PA1 PHI PTH RPD SET STY TIH TRG VGI FOE GAA GLB GN7 HSM HV5 IPT LTR MAT MGA MLZ NBG NVA PAO PHL PYA RPL SFG SVA TMB TRN X FTR GAM GLD GPM HAR IAS ITR LYM MBG MGL MNV NED OCY PAQ PHM PYX SBD SGA T6A TMD TRO X2F FTY GAP GLF GS HGA IDG IYG LYZ MCY MGN MRP NEP OIC PBB PNA QSI SBL SGC T6P TMP TS YOF G16 GC4 GLM GSA HIC IDS TYR M3L MDA MGY MSA NFA OIP PDU PNG QUO SCH SHP TAF TMR TYI YYG GIP GCM GLP GSC HIP IDU KAI M6P MDM MHL MTY NGA OMP PEC POM R SCR SLZ TBG TNB TYN G
69. Release Notes 10 Fixed a problem with reading spreadsheet information in state files generated by VIDA 2 0 x which caused extra tabs to appear in the spreadsheet Fixed a problem with writing spreadsheet information correctly in VIDA 2 1 Fixed a crash when reading a badly formatted state file potentially generated by the bug fixed above Fixed acrash when attempting to import a non standard whitespace delimited spreadsheet whites pace is not a supported spreadsheet delimiter Fixed a crash on certain video cards when attempting to display fixed size text labels Fixed a crash caused by dragging state files onto the application icon in order to open them Fixed an application hang when reading badly formatted XPLOR maps Fixed an application hang when reading a badly formatted OEB file Fixed a memory leak generated when running a large number of scripting commands Fixed a crash when trying to generate electrostatic contours on extremely small molecules e g water Improvements 10 11 12 Filled out Sybyl mouse map in more detail Added fixed size text option for labels see Preferences such that the label font size remains fixed independent of zooming in the scene however the font size can be adjusted using the Text Scale slider in the Style Bar window Added the ability to specify the font for labels when using nice fonts for labels see Preferences Added
70. TDD ITLL OY SWNT MVE RREY RUVEGAMS SYFAPDLILNEQRMKESSE LOMKVLLLINTI PLEGLRSOTOF EEMRS SY IRELTKATGLRORGVVSSSQRFYOLT ILVKOLHEYCINTEIOSRALSVEF PEMD LIPPLINLIMSTEPDVIYACHONTREDTSSSLLTSINOLGEROLLSVVEWSKSLPGERNLHIDDOTTLIOY SuIMSIMVE GLGWRSY KHVSGCMLYE APDLILNEQRMKESSE 5 YSLCTTMNOT POEEVKTOVSOEEF LOMKVLLLINTI PLEGERSOTOR EEMRS SYTRELIKATGLROKGVVSSSORFYOLTKLLINLHDLVKOLH YCINTF TQSRALSVEF FEM p10 SEVIAAQLPKTLAGMVKELLFI Scripting Window CC O COH CACOOH COD NCTCOHCOHZECCI CC O CC COTA Figure 3 2 Docked window layout The layout including the position and visibility of the main and peripheral windows is saved on exit and will be restored the next time the application is run The layout of the windows can be changed by clicking on the title bar of the individual window and dragging it to the desired location A placeholder window will appear when the mouse is over an area where the dragged window can be placed as seen in Figure 3 3 Furthermore multiple windows can be tiled on top of each other into a tab controlled area as can be seen in the bottom window in both Figures 3 2 and 3 3 In addition to manually changing the layout there are a number of predefined layouts which are acces sible via a drop down button on the toolbar as can be seen in Figure 3 4 The colored icon associated with each option indicates which peripheral windows will be shown and where Hovering the mouse over any given option for about one second will generate a preview
71. Zoom The scene can be scaled or zoomed by holding down the Middle mouse button the Left and Right mouse buttons together or by using the mouse wheel Any of these three modes will scale the scene Multiple modes are provided for convenience and to accommodate the many varied mouse configurations in existence The scene can also be scaled using the W and S keys on the keyboard 36 Chapter 5 3D Display Text Scale The scale of the text displayed in the scene can be controlled by using the mouse wheel while holding down the Ctrl key Clipping The position of the near and far clipping or slabbing planes can be controlled using the mouse wheel while holding down both the Ctrl and Shift keys Both planes are moved simultaneously and mirror each others positions It is important to note that slabbing must already be enabled for these operations to actually be performed Contour Level The contour level of the grids in the default scope can be adjusted using the mouse wheel while simul taneously holding down the Shift key Each incremental turn on the mouse wheel corresponds to an increase or decrease in the contour level by 0 1 Label Informative labels can be displayed about atoms and bonds as well as surfaces and grid contours un derlying the current mouse position if the Ctrl key is pressed while moving the mouse no mouse button need be depressed on most platforms On the Mac this behavior is obtained by holding down t
72. a custom option to the atom and bond label dialogs to allow for easier specification of custom labels without having to use scripting commands Improved the drawing of labels such that they always appear in front of the object they are labeling Improved the ability to see the selected state of partially selected residues in the sequence view Added file dialog filter option to allow reading of ASCII formatted grids agd Added support for native builds on Intel Macs Added interactive text annotation of molecules Added the ability to start monitor terminate and load results from external processes Added a protein sequence viewer as part of the 2D depiction widget Added the cechemlite Python module enabling significantly greater scripting functionality within VIDA 21 5 Version 1 2 0 149 13 14 15 16 17 18 Added the ability to create arbitrary sphere monitors via the scripting API see Python API for documentation of the new method CreateSphereMonitor Added support for fullscreen display Added new cloud visualization style for grids Added support for copy and paste of molecules from ISIS Draw Added support for use of the Maestro mouse map in the 3D display Added support for saving screenshots in JPG format in addition to PNG 215 Version 1 2 0 January 2006 Minor Bug Fixes Fixed a number of preferences that were not being properly saved or obeyed by the application Fixed the un
73. a cell in the spreadsheet will cause the associated molecule to become the Focused molecule see Section 3 3 1 Double clicking on a cell in the spreadsheet enables modification of the data value in that particular cell Some columns cannot be changed such as the VIDA ID or Depic tion columns Clicking on a cell and the holding the mouse button down while dragging selects a rectangular region in the spreadsheet This region can be copied and pasted into other spreadsheets The depiction column will be automatically converted to a SMILES string when feasible molecule has fewer than 100 atoms Similarly clicking the mouse on a molecule s depiction and dragging the mouse to another application will transfer that image to the other application This provides an easy way to transfer molecular images to other documents Clicking on a cell in the property columns Visible Marked or Locked will toggle the respective property To assign a property to a selected range of cells simply select the range and then right click on the spreadsheet and select the appropriate action from the popup menu 11 2 Sorting A spreadsheet can be sorted by clicking on a column header That spreadsheet will be sorted according to the data values in the associated column If the data in the column to be sorted contains both numeric and non numeric values each set numeric and non numeric will be sorted independently and then concatenated together Multiple
74. ace will select the entire surface 5 12 Symmetry 65 5 12 Symmetry AFITT supports symmetry operations This support includes a menu under the Edit menu see 5 13 which controls displaying symmetry in the 3D window and some items in the popup menu of the list window see 6 4 which allow setting or editing symmetry parameters Symmetry information may be read from certain files When AFITT reads PDB format files which have valid CRYST1 records symmetry information is taken from that record for all molecules read from that file Similarly when AFITT reads MTZ format reflection files the symmetry information is read from that file and of course AFITT will read symmetry information if it is present in an oeb file As described in 5 13 when symmetry display is enabled all symmetry operators which would result in a molecule within a certain distance the symmetry radius of the screen center are displayed This means that as the view is translated certain symmetry replicates will appear and disappear as they move into or out of the symmetry zone There is therefore no need to manually re calculate the symmmetry after translating the model 5 13 Symmetry menu The symmetry menu is located in the Style menubar The items in the symmetry menu are e Edit cell This item allows editing or setting of the unit cell and space group parameters of the active object which will generally be either a reflection data set or a molecule In addition
75. ake that object the Focused object see Section 3 3 1 In the List Window the Focused object s name is displayed in bolded text and there will be a small blue box around the dot in the second column the Visible column An example of this can be seen in Figure 12 1 where 12 4 List Columns 109 List Window iJ List Window 1 1 cox2 1 3 cox2 1 4 cox2 1 5 cox2 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 10 cox2 1 16 cox2 1 17 cox2 1 18 cox2 1 19 cox2 EE E3 GB a E H HAE 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 1 29 cox2 1 30 cox2 5 i 7 16a cox2 1 31 cox2 z L 7 17a cox2 1 32 cox2 5 1 33 cox2 1 34 cox2 1 35 cox2 1 36 cox2 1 37 cox2 E y HAE amp G ee a Flat view b Spreadsheet view Figure 12 2 Alternate views the molecule 1 1 cox2 is Focused The second column is the Visible column The Visible column has a green dot icon in its column header This column displays and controls the Visible property for objects If an object is Visible there will be a green dot identical to the one in the column header present in that object s Visible column If that object is also the Focused object there will be a small blue box around the dot in that column as well If a given object has children the visibility of its children can also be controlled in this column Child objects have an additional layer of control regarding visibility in that they can be hid
76. any time and are fully undoable using AFITT s undo operations available in the Edit menu AFITT divides crystallographic workflow into multiple Tasks Each task has a seperate set of user interface widgets and components designed to facilitate workflow The current tasks are Ligand Protein and Refinement Each Task has a different default layout that may be changed by the user For each task however there are commonalities that will be discussed first 3 3 Object States AFITT is a stateful application and employs many special states or properties in its interaction with the user The most important of these are Focused Visible Locked Marked and Selected Each object displayed on the screen can be in one these states By object we mean anything loaded into AFITT including molecules density maps and surfaces sometimes refered to as blobs Some of these states are persistent such as the Locked state and some may be transient for example a Focused object will be replaced when a new object becomes Focused The main reason that states are important is that many operations performed in AFITT operate on a subset of objects and this subset is determined by a given objects state The user can choose a default scope of operation For example if the user wants to make a molecular surface for every loaded molecule the A scope should be chosen For more information on scope see section 3 4 3 3 1 Focused State The Focused state is a pr
77. applied to all of the objects in the current scope The second button with the molecule overlayed on a color wheel prompts the user to select a specific color that will be applied to all molecules in the current scope In addition this button contains a drop down menu of molecule specific coloring schemes which can be applied Detailed information about the individual color schemes can be found in Section 5 9 2 The third button with the surface overlayed on a color wheel prompts the user to select a specific color that will be applied to all surfaces in the current scope In addition this button contains a drop down menu of surface specific coloring schemes which can also be applied Detailed information about the individual color schemes can be found in Section 5 11 2 The fourth button with the grid overlayed on a color wheel prompts the user to select a color that will be applied to all grid contours in the current scope The fifth button with the U icon overlayed on a color wheel assigns a unique color to every object in the current scope The last button restores the original color of all the objects in the current scope More details about the specific coloring schemes can be found below Beneath the buttons is a slider which can be used to adjust the transparency of surfaces see Figure 5 2 a and grid contours The slider values range from 0 completely opaque to 100 completely transparent The specified transparency value is appl
78. association of unrelated Python scripts For more details about Python scripting please see Chapter 14 as well as the associated Python scripting API documentation 4 2 Closing Files 27 4 1 6 State The entire state of a session can be stored in a single file called a State File oes A state file contains all of the molecules grids and surfaces that were loaded in AFITT at the time the state file was created In addition the state file preserves the actual view layout and properties of that session State files provide an extremely convenient way to save sessions for later work or to share with collaborators Furthermore state files are the fastest method of reading and writing large data sets To load a state file choose the Open State option in the File menu and select the desired state file State files can also be loaded from the commandline It is important to note that loading a state file during a run will clear the current state before loading the new one Therefore be sure to save the current state before loading a new state if keeping the current state is desired Furthermore since state files contain the entire state of the application that generated them loading a state file will overwrite local preferences however they will not be saved on exit 4 2 Closing Files When a file is opened in AFITT its contents are loaded into its own individual list To close a given file simply right click on the assoc
79. aste option can be found in the Edit menu and can also be performed by pressing Ctrl V on the keyboard This will paste any molecules found on the clipboard into a new list called Pasted CHAPTER FIVE 3D Display The 3D display is the primary visualization interface available to the user although it is not the only one The purpose of the 3D display is to allow the user to view and interact with molecules see Section 5 9 grids see Section 5 10 and surfaces see Section 5 11 5 1 User Interaction The primary interface mechanism to the 3D display is the mouse which is discussed in detail in Section 5 1 1 However there are a large number of potential operations that can be performed in the 3D display which necessitates an additional mechanism of interaction besides the simple use of mouse buttons and popup menus For this reason a separate peripheral window see Section 3 5 2 is provided to access these operations This window is called the Style Control and is discussed below in Section 5 1 2 5 1 1 Mouse As described above the mouse is considered to be the primary interface mechanism to the 3D display The details of how to use the mouse to interact with the 3D display are listed here A three or two button mouse is recommended for use however support for a single button mouse is available Holding down the Ctrl key while clicking or pressing the mouse button will emulate using the Right mouse button on a three bu
80. at WAT H20 HOH TIP SOL e r Ssubstrate this is defined as r wat amp amp r Saa amp amp r dna There are two atom name macros as well a backbone which may be abbreviated as a ba which matches specifically protein backbone atoms and a sidechain macro a sch which matches protein sidechain atoms Both of these can be negated a Sbackbone or a S sch 15 6 Scripting with ScratchScope In addition to the built in commands Visible Select Lock Mark and Subset it s quite straight forward to use a selection string with any command which operates on a scope see 3 4 by binding the selection string to the ScratchScope For example the following function defines an atom coloring command ac def ac color str Subset scratch str use string to make named subsett ScratchSet scratch bind it to scratch scope AtomColorSetScoped OEggColor color ScratchScope use it With two small helper functions these types of functions can be made even easier to create def ScratchSubset str Subset scratch str ScratchSet scratch return ScratchScope def ScratchSubsetList str Subset scratch str 15 6 Scripting with ScratchScope 127 ScratchSet scratch return ScratchlList With these functions defined the ac function above and functions similar to it can be easily defined For example set atom color def ac color str AtomColorSetScoped color ScratchSubset str
81. attempt to fit to them The system can also wait for new data to appear The automation is enabled from the command line by sending the e automate flag to afitt Every other command line parameter is then passed to the automation system 19 1 Getting Started With Automation The 1115 protein and ligand shipped in the DATA directory will be used as an example That assumption here is that AFITT is being run from the installation directory The examples given are intended to gently walk you through the automation system and a full list of options are included at the end of this chapter By default a filenamed results sdf is output in the working directory Note that the working directory may change during an automation run see Section 19 2 1 If a file with the same name already exists in the working directory a new one will be created with a number appeneded For example if result sdf already exists a new file results_1 sdf will be created 135 136 Chapter 19 Automation To stop an automation run simply click on the Stop icon by the progressbar Note that it may take a minute or two to stop a run 19 1 1 Fitting a Single Ligand For example to fit the ligand 5 to density bin afitt e automate grid DATA 1115 grd ligand DATA 1115 lig pdb gz protein DATA 1115 nolig pdb gz Notice that after the fitting is done AFITT appears to hang This is because AFITT is waiting for new data to appear For
82. ays masks visible molecules that are on screen during this procedure If a blob is picked that has a ligand inside of it then the blob in actually will be masked away For best results hide any molecules that are not modeled 7 7 Working With Poor Density 79 7 73 Add a Box Constraint Constraint regions can be added to limit the areas in which blobs can be detected A constrain region is a bounding box or volume of space where blobs are allowed to be located The box itself can also be used as a density blob Boxes are defined by molecules or atom selections on a molecule This is done to simplify box creation A box can be generated from a ligand or select some atoms on a protein to generate a box To add a box simply select the Task Add Box menu option from the top menu This will prompt for a molecule id and then a padding amount When complete the bounding region will be displayed in the 3D window This region will also be added as a blob to the blob window which can immediately be used as a blob for fitting purposes The next simplest way is to select some atoms that form the boundary of the desired region For example if you have a protein loaded and you can select two atoms that are on the boundary of the pocket Once the atoms are selected you can choose the Add Box From Selected option from the Task menu Boxes can also be imported from any molecular file format For example an xyz formatted file is as follows 2
83. be one of the ones listed in the Writing Refinement Dictionaries in chapter 7 8 as these may conflict with the standard refinement dictionaries shipped with refmac or cns x 21 2 Version 1 3 0 August 2007 Minor Bug Fixes 1 Fixed a problem where on some graphics cards only aromatic rings would be drawn when a molecule was in stick mode Major Bug Fixes 1 2 Upgraded to OEChem 1 5 which fixed a number of bugs Upgraded Omega Toolkit to 2 2 1 Improvements 1 2 3 All modeling widgets have been overhauled Blob finding is heavily improved in speed and accuracy Fitting information is now displayed in 3D window 4 Automated protein clean up is now available 144 Chapter 21 Release Notes 10 11 12 13 14 15 16 17 18 Modeling mode is now removed Internal conformation generation is now consistent with Omega 2 2 1 Added Coot mouse mode Anti aliasing of text and 2D depictions Much better control over placement of dock windows Dock windows can be stacked on top of each other in tabs Single toolbar at the top of the application Additional popup toolbars placed around edge of main window Enabled grid contour transparency Hydrogen style is now a molecule specific property instead of a global property C Alpha trace display is now a molecule specific property instead of a global property Ribbon display is now a molecule specific property instead of a global prope
84. board different from the system clipboard may then be used for subsequent Paste operations e Cut This command similar to Copy places a copy of the currently modeled atoms on the clip board Unlike Copy however the atoms are then deleted from the current molecules If deleting the atoms involves breaking bonds the implicit hydrogen count is adjusted e Paste Add the contents of the clipboard to the each currently modeled molecule The entire clip board will be added to each molecule even if the clipboard consists of parts of multiple molecules Care should therefore be exercised when calling this function if multiple molecules are being mod eled simultaneously e Paste special This menu contains one or more special options for pasting 6 4 Popup menus 69 The Paste special menu contains e Alternate conformation This option allows pasting the contents of the cut buffer into a PDB style alternate conformation i e it sets the OEChem residue s AlternateLocation property The user will be prompted for an alternate location identifier a one letter code used to distinguish alternate conformers the occupancy of the new atoms and to determine if any backbone atoms should be pasted into the molecule Atoms on the clipboard are pasted into the modeled molecule s and the occupancy and alternate location property are set appropriately At present the occupancy of atoms already in the model is not changed Splitting
85. cated Modeling functionality includes bond operations such as breaking and creating bonds residue operations such as creating or replacing residues and rotamer operations such as locating the highest probabillity rotamers from the various rotamer libraries A break down of the modeling operations can be seen in Figures 3 8 3 9 and 3 10 e Mouse Toolbar this allows control over modifying coordinates torsions and rotatable bonds as well as inspecting atomic properties such as dihedral angles and residue information This toolbar is in the right hand side of the 3D window Chapter 3 Getting Started Break Bond Determine Bonds OJO Sacrifice Fly Double Single Create Bond Figure 3 10 Bond Controls Mouse mode operations change the behavior of the mouse in the 3D window The default behavior of the mouse controls selection and rotation of the main view Other mouse modes control altering residue and bond torsions translating the modeled atoms in 3D and modifying and placing the c alpha backbones Holding the mouse over each button reveals a tooltip describing the operation that will be per formed Toggle Auto Hide Toolbar Mouse Performs Selection Default Mode Shortcut w Mouse Performs Identification Measure Distances Measure Angles Measure Torsions Translate Selection Shortcut t Rotate Around Selection Center Shortcut Shift R Rotate Around CA Sh
86. ce is com puted by an average distance from the blob to the currently modeled protein By sliding the distance slider seen in Figure 3 6 or by typing in a value in the text entry the default distance can be altered 3 12 Ligand Task 15 Ligand Mode Data Data Visibility Click to Focus Clear Data Browse For Data Model Ligar d ccp m ap lafq ccp 92 Hide Show Auto Find Density Modeled Density Search Density Current Threshold For Ligand Sized Distance h Regions 103 3745 3 2300 39 7700 3 3600 Select For A Fitting Control 3 3900 Blob List 4 0500 4 3100 Visibility Control ID and Blob Volume and Contour level Distance to protein for blob selection Fit Ligand To di ae Checked Blobs Toggle conformer generation Mask Map To Checked Blobs Picked Blobs from 3D Window Merge Checked Blobs Delete Checked Blobs Reset 3D View to Modeled Data Figure 3 6 Modeling Ligand Control 16 Chapter 3 Getting Started Finally once one or more blobs has been selected click on the Fit button to begin the fitting process When complete the fitted small molecules will appear on the screen and in the spreadsheet 3 12 2 Analyzing Ligand Results AFITT fits ligands to density using a combined forcefield that forces ligands into density while main taining appropriate chemistries The potential function being used to adapt the ligand is V Vis ae AVshape where V represents the internal energy of t
87. certain residues the selection slider seen in Chapter 5 11 3 can be used to filter out ligand density that is not close enough to the protein Simply select the residue by double clicking then adjust the selection slider or type in a number in the widget Then when next clicking on the Find Blobs button any blob that is not within the desired distance will be rejected 7 7 Working With Poor Density In some cases the appropriate ligand density will not be found usually because the density is too noisy When this happens there are several options both automatic and manual 7 7 1 Split the Protein Sometimes the protein has ligands embedded in the structure Because all components of the protein are used to reject non ligand dentisy AFITT can be fooled into rejecting density where the ligand has already been placed Note Molecules may be split using the New Molecule From Split option located in the File menu to separate the ligand waters and fragments from the protein 7 7 2 Manual Blob Picking The blob picking button located in the Ligand Model window places AFITT in blob picking mode When a piece of density is clicked on in the 3D window AFITT will attempt to extract the isocontour into a density blob In many cases the extracted density can be larger than what is desired however the scribing tools located in the style menu can be used to crop the surface to the correct size see Chapter 5 11 for more details Note AFITT alw
88. ch as expected is interpreted as a single molecule However sometimes the ability to interact with the individual components is of particular value The ability to split a molecule into separate components can be performed by selecting the Components option in the From Split submenu in the New Molecule submenu in the top level File menu This function will then determine what the individual components of the Focused molecule are and create a new list containing a new molecule for each component The 5 9 Molecular Visualization 55 a Wireframe b C Alpha Trace c Ribbon Figure 5 13 Different views of the same protein a Protein with Waters b Protein without Waters Figure 5 14 Same protein with and without waters a Multimer displayed as a single b Multimer displayed as multiple molecule molecules Figure 5 15 Same protein as a single molecule vs as multiple molecules original molecule is not changed or deleted An example of this process can be seen in Figure 5 15 The From Split sabmenu also contains Selected and Marked options which will create new molecules from the original based on the selected or marked set of atoms It may also be of interest to put some of these components back together into a single molecule This can be done by choosing one of the following options from the From Merge submenu in the New Molecule 56 Chapter 5 3D Display in the top level File menu Selected
89. clude Microsoft Windows Linux SGI Irix and Mac OS X 2 1 Microsoft Windows A simple installation wizard is provided for Microsoft Windows Double clicking on the file e g afitt 1 3 microsoft win32 1686 setup exe will start the wizard which will direct the installation process It is recommended that the installer to be allowed to install AFITT 1 3 in the C OpenEye directory if possible Once the installation process is complete there will be a AFITT icon on the desktop as well as in the start menu To uninstall AFITT simply click on the Uninstall AFITT icon in the C OpenEye directory or in the start menu in the OpenEye AFITT group AFITT can also be uninstalled using the Add Remove Programs option in the Control Panel 2 2 UNIX Linux The UNIX Linux installation is provided as a gzipped tarball e g afitt 1 3 redhat 9 0 i586 tar gz The first step is to create the directory where AFITT will be installed e g usr local openeye Next move the installation file to that directory and then gunzip and untar the file using the following commands tar zxvf afitt 1 3 redhat 9 0 1586 tar gz This will create a AFITT directory with all of the appropriate files in it To run AFITT enter this directory and call afitt This directory can also be added to the PATH environment variable so that af itt can be typed anywhere 2 2 3 Mac OS X 3 to launch the application To uninstall AFITT simply remove the directory in wh
90. columns can be sorted simultaneously by selecting the Sort Spreadsheet option from the top level Data menu This will launch a dialog that can be seen in Figure 11 2 This dialog can also be launched by right clicking on any column header and selecting the Sort Spreadsheet option from the generated popup menu Spreadsheet Sort Ascending O Descending Move Sorted Columns to Start Figure 11 2 Sorting the spreadsheet This sorting dialog allows for the selection of up to three columns The spreadsheet will be sorted by the first column selected with ties being broken by the remaining columns The sorted columns can be moved to the front of the spreadsheet by selecting the Move Sorted Columns to Start option in the dialog The first time the spreadsheet is sorted it may take longer than expected as the data values need to be retrieved from the molecule and any expressions must be calculated The progress of the sort is indicated 98 Chapter 11 Spreadsheet in the progress bar at the bottom of the application If desired the sort can be aborted by clicking on the stop button to the left of the progress bar 11 3 Displaying Data The visibility of columns can be toggled by selecting the associated column name from the right click popup menu generated by clicking on any column header Data column names can be found in the Column Visibility submenu Hiding the depiction column can allow
91. d File Prompt button to the right of text entry box More details on adding prompts can be found in Section 13 1 3 If the desired file will need to be created each time it is possible to create the desired file at start time by writing out selected molecules or grids directly from AFITT To use this feature click on the Add Input button to the right of the text entry box At start time the user will be prompted to select the desired molecules or grids which will then be saved to a file which will be passed as a command line argument to the process For more details on adding an input file see Section 13 1 1 In addition to being able to specify specific input files it is possible to specify an output file which can potentially be read back into AFITT when the process is completed As discussed above it is possible to a specify specific filename in the command line or to use a prompt but if it desired to load the resulting file the Add Output button should be clicked to specify the file For more details on adding an output file see Section 13 1 2 Besides being able to specify filenames it is often desirable to be able to specify other variable arguments at start time To do this click on the Add Text Prompt button which will add a prompt argument to the command line arguments At start time the user will be asked to specify the desired text to be passed to the process For more details on text prompts see Section 13 1 3
92. d license provided by OpenEye must be present on the installed computer OpenEye license files are typically named oe _license txt and most OpenEye software products 4 Chapter 2 Installation including AFITT expect to find that license file in the location specified by the OE_LICENSE environ ment variable or in the directory specified by the OE_DIR environment variable If neither of the above environment variables are defined on the computer AFITT will prompt the user at run time to locate a valid license file If a valid license file is specified AFITT will remember the location of that file and will not prompt again until that license expires or is removed CHAPTER THREE Getting Started This chapter is designed to help familiarize the user with the AFITT application and interface in general While AFITT looks very similar on all platforms there are subtle differences between platforms such as how each platform displays certain specific dialogs the color selection dialog for instance All screenshots provided in this manual were taken using the Microsoft Windows version of AFITT and therefore the actual appearance on other platforms may vary slightly from the images seen here 3 1 Why Another Modelling Program Current methods of ligand fitting such as topological analysis of electron density 1 global optimization of position and conformation of a ligand in a density blob 2 interatomic distance matrices 3 4 5 6 7
93. den visible or can mimic its parent s visibility If an object is set to mimic s its parents visibility an upright triangle is shown instead of the standard green dot as can be seen in Figure 12 3 Clicking in this column cycles through these three states in the following order hidden mimic visible Please note that molecular structure atoms residues chains etc are not considered children objects and as such always mimic the parent molecule s visibility if they are marked visible with a green dot The third column is the Locked column The Locked column has a small grey padlock icon in its header If an object is Locked there will be a grey padlock identical to the one in the column header present in that object s Locked column The fourth column is the Marked column The Marked column has a red check mark icon in its header If an object is Marked there will be a red check mark identical to the one in the column header present in that object s Marked column The fifth and final column is the JD column An object s ID is simply a unique identifier that can be 110 Chapter 12 List Management List Window Name a7 cox2_good sdf gz D 5 E 1 1 cox2 GH Structure Molecular surface 1 3 cox2 1 4 cox2 1 5 cox2 1 6 cox2 1 7 cox2 1 8 cox2 1 9 cox2 1 10 cox2 1 16 cox2 1 17 cox2 1 18 cox2 1 19 cox2 1 20 cox2 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 1 29 cox2 1 30 cox2 1 31 cox2 1 32 cox2 pa
94. ding down the Shift key when selecting will allow selection of a continuous range of residues The current selection is reflected in the coloring of the 2D depiction There may be slight variations between the depicted selection in 2D and 3D due to the difference in how hydrogens are drawn CHAPTER ELEVEN Spreadsheet The spreadsheet window is used to view compute and analyze data associated with molecules and can be seen in Figure 11 1 The spreadsheet window contains three initial spreadsheets by default Molecules Proteins and Atoms The Atoms spreadsheet is slightly different in behavior from the other two and is described later The Molecules spreadsheet is populated with all currently loaded small molecules fewer than 255 atoms Conversely the Proteins spreadsheet is populated all currenty loaded molecules that have more than 255 atoms The columns and their associated values are populated from SD data SD data can be found in both SDF and OEB files Additional spreadsheet columns can also be added by computing expressions see Section 11 5 or by importing external data from a comma seperated value csv or tab seperated value file txt see Section 11 9 11 1 Basic Usage Columns are divided into two basic types data and property Data columns are those found in SD data and or imported files Property columns include e Depiction This column contains an image of 2D structure of the associated molecule e VIDA ID This
95. do stack such the initial setup of the 3D display cannot be undone by accident Fixed the display of angle monitor measurements from radians to degrees Fixed the display of the text scale widget such that it is not hidden on low resolution monitors Made sure that when saving images and state files a proper file extension is added to the filename even if one was not specified Major Bug Fixes 1 Fixed a crash when trying to generate electrostatic contours on extremely small molecules e g water Features Overhauled AFITT s interaction model to be sane Added interactive text annotation of molecules Added the ability to start monitor terminate and load results from external processes Added a protein sequence viewer as part of the 2D depiction widget Added the oechemlite Python module enabling significantly greater scripting functionality within VIDA 150 Chapter 21 Release Notes 9 10 11 Added the ability to create arbitrary sphere monitors via the scripting API see Python API for documentation of the new method CreateSphereMonitor Added support for fullscreen display Added new cloud visualization style for grids Added support for copy and paste of molecules from ISIS Draw Added support for use of the Maestro mouse map in the 3D display Added support for saving screenshots in JPG format in addition to PNG Improvements 1 2 Cleaned up the appearance of th
96. dual processes into a larger process The Process Manager contains two tabbed windows called Processes and Status respectively as can be seen in Figures 13 1 and 13 2 The Processes tab contains the list of available processes and the functionality to create and edit them The Status tab displays all of the currently active and recently completed processes and information about their state It also displays the commandline output generated by each of the processes Beneath the list of processes in the Processes tab is a collection of four buttons The button on the left with the play icon starts the currently selected process The next button with the plus icon clears the editor on the right hand side to allow for creation of a new process The next button with the link icon changes the editor on the right hand side to allow for creation of a new pipe see Figure 13 6 The last button with the delete icon deletes the current selected set of processes from the list of available processes Process Manager Processes Status Omega Rocs Name Omega Executable C cygwin usr local openeye bin omega2 exe Browse Rocs Pipe in lt input_d molecule oeb gt out lt output_d oeb open gt fraglib Add Input C cyqwin usrjlocalfopeneye data omega2 fraglib oeb gz Add Output Add Text Prompt Add File Prompt O ana ma Figure 13 1 Process Manager 113 114 Chapter 13 Process Management
97. due information associated with a given vertex This scheme will not work if the surface was not created from a protein and also if that protein is not currently present There are four different scales available Charifson Eisenberg Kyte Dolittle and White Octanol The Charifson scale applies defined colors based on residue information The other three scales each return a specific hydrophobicity value which is then used to return a color based on a brown to blue gradient between 2 5 and 2 5 for Eisenberg 4 5 and 4 5 for Kyte Dolittle and 4 0 and 4 0 for White Octanol Charifson 5 11 Surface Visualization 63 All ASP GLU ASN GLN HIS LYS ARG ALA PHE ILE GLY LEU PRO VAL MET CYS CYS TRP TRP SER SER THR THR TYR TYR Other CA CB CG CD Yellow Red Red Magenta Magenta Magenta Blue Blue Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Magenta Yellow Magenta Yellow Magenta Yellow Magenta Yellow Magenta Yellow White 0 255 255 255 0 0 255 0 0 192 0 192 192 0 192 192 0 192 0 0 255 0 0 255 255 255 0 255 255 0 255 255 0 255 255 0 255 255 0 255 255 0 255 255 0 255 255 0 192 0 192 255 255 0 192 0 192 255 255 0 192 0 192 255 255 0 192 0 192 255 255 0 192 0 192 255 255 0 255 255 255 64 Chapter 5 3D Display Eisenberg Kyte Dolittle White Octanol
98. e Tiled mode can be toggled on or off from the main application toolbar When the 3D display is in Tiled mode every Visible and Focused object is displayed centered in its own individual pane However if desired each object can be shown relative to the same center if the Auto Center Panes option in the 3D Display section of the application preferences is unchecked It is important to note that when Auto Center Panes is enabled it is not possible to recenter or translate using mouse interactions in any of the panes To indicate which object is Focused see Section 3 3 1 the corresponding pane will contain a blue border just inside the regular pane border If there are any Locked objects see Section 3 3 3 each of them will be displayed in every pane in addition to the individual Visible and Focused objects 5 7 Toolbars The 3D display provides additional toolbar areas aside from the main application toolbar inside the 3D window There are four possible toolbar locations at the top of the window at the bottom of the window at the left edge of the window and at the right edge of the window Toolbars in these locations are hidden by default but popup when the mouse is moved over the toolbar area and disappear when the mouse leaves These toolbars can be made sticky by clicking on the button with icon in it 46 Chapter 5 3D Display By default the 3D display provides a Style toolbar at the top of the
99. e Chapter 11 7 4 2D Preview Located at the bottom right this shows either a 2D depiction of the small molecule or the sequence of the protein depending on which one is currently focused 7 5 Locating ligand density AFITT locates density suitable for ligand placement based on both the type of map being analyzed and the volume of the ligand to be fit In the cases where a conformation is given AFITT computes the volume of the supplied conformation otherwise the volume of the conformation is estimated through a simple linear approximation based on the number of atoms in the ligand Given the estimated volume of the ligand AFITT starts from the maximum amplitude of the density map and ramps the contour level down until blobs are found within a range of the ligand volume If the discovered blobs are small relative to the ligand AFITT includes nearby blobs in an effort to try and match the ligand AFITT attempts to include regions within a certain distance from the modeled protein if available by default however based on the orientation of the protein the search range may include solvent density or symmetry related blobs that are not near enough to the protein 74 Chapter 7 Ligand Modeling Because of this AFITT includes a distance slider to prune blobs within a certain distance to the protein The default distance is 44 7 6 Filtering Ligand Density Based on Distance to a residue If the ligand is known to be interacting with
100. e at start time before the Python function is called Finally there is a checkbox at the bottom of the dialog to specify whether or not this is a default input The default input entry is used when constructing pipes and will be discussed in Section 13 2 13 1 2 Adding Output The Output Editor allows the user to specify a mechanism by which an output file of the process to be run is handled by AFITT At the top of the dialog are two mutually exclusive options the first is the Temporary check box which indicates that the output file is a temporary file and will be removed after it has been handled by AFITT The other option is a field which allows specification of a specific filename to be used for the output file If this option is selected the file will not be deleted after it is handled 116 Chapter 13 Process Management EN Output Editor Filename Browse Ignore Python Default Output Figure 13 4 Process Output Editor Once the above has been specified the user can specify how AFITT should handle the file when the process is finished There are three possible options the first is to open the file the second is to ignore the file and the last is to call a Python function to handle the resulting file If it is desired to open the file the file format that the process should use to generate the output file must be specified so that AFITT will know how to properly load the results If a Python function
101. e of operations select the Mouse Performs Selection option from the Mouse Toolbar seen in figure 3 11 As seen in Figure 3 7 the Protein Mode task is divided into sections 18 Chapter 3 Getting Started File Edt Task Data Style View Window Help Focused Visible Marked ai a mI List Window ax El Style Name 02 mp Modeling g A28 in EN El a a LI Ea E put pdb 9 x EJ z a e a e 5 1428 pdb ligand 6 2 1428_afitt_lig pdb 7 al 1420 08 end PR e tbn tbldtdllB ts tb 10000000080 ES 10 10 99 999 9 ca Regular Map 2Fwt 1428_prot mtz Difference Map None Color er Gade Transparency g T x B E BEA Molecules gt i 7 NE az X Ramachandran Richardson General QLI PPLINLIMSIE PDVIYAGHDNTKEDT SSSLLTSLNOLGERQLLSVVEWSKSLPGERNLHIDDOITLI Gy SWwMSLMVE GOGWRSYKHVSGOMLYFAPDLILNEORMKE SSE a 5 O POPE VETS P EEVEE EEE LOMEVLLLINTL PLEGLRSOTOFEEMRSSYIRELIKAIGLROKGVVSSSORFYOLTKLLONLHDLVKOLHLYCINTFIQSRALSVEF PEM LIPPLINLIMSIEPDVIYACHONTKEDTSSSLLTSINOLGEROLLSVVKNSKSLPEGFRNLHIDDOITLIQY SWMSLMVE GLGWRSYKHVSGOMLYF AEDLILNEORMKESSE 85 SLOT POEEVELOVSCEEFLCMKVLLLINTI BLE GLRSOTOF EEMRS ST RELIKALGLROWVVSSSORE YOLTRLLENLHDLVRQLHLY CINTELOSRALSVEE FEM b10 SEVIAAOLPRT 2D Preview Scripting Window CC 0 C H 1CC C H 2 C 1 CC C H 3 C H 2ccc4 CC Oj CC C 34C C Figure 3 7 Protein mode layout e List Window On the left side the Lis
102. e right click popup menus Added a Style option to the right click popup menus when appropriate to the context which mirrors the main Style menu Added significantly greater detail to info labels created when holding down the ctrl or alt on Mac key when moving the mouse in the 3D or 2D views Added two default filtered spreadsheets to the application Molecules and Proteins Filled out the scripting API for greater control of the GUI in particular for menus and toolbar Greatly improved the electrostatic coloring scheme for protein surfaces Added the ability to change the font used by the application 1 2 3 4 5 6 7 _ 8 a 9 10 11 BIBLIOGRAPHY Menend z Velazquez A Garci Granda S A procedure towards the automatic solution of crys tal structures by means of topological analysis of Fourier maps J Appl Cryst Vol 36 pp 193 205 Diller D J Verlinde C A critical evaluation of several global optimization algorithms for the purpose of molecular docking J Comp Chem Vol 20 1740 1751 1999 Koch M H Automatic interpretation of electron density maps for organic structures Acta Cryst A Vol 30 pp 67 70 1974 Main P Hull S The recognition of molecular fragments in E maps and electron density maps Acta Cryst A Vol 34 pp 353 361 1978 Cascarano G Giacovazzo C Camalli M
103. e same name already exists in the directory a new one will be created with a number appeneded For example if result sdf already exists a new file results_1 sdf will be created 138 Chapter 19 Automation 19 2 3 Testing the Data Without Fitting For complicated searches the data that will be used can be tested without actually performing the fit by using the testonly option This does everything except the actual fitting process and can be used to see if the command line is set up correctly For example bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit testonly 19 2 4 Adding Box Files Box files can be added by using the boxes switch This switch is expanded the same way as the ligands are Each box found identifies a unique region that blobs must intersect in order to be used for fitting This is a convenient way of defining a receptor site and pruning out regions that should not be used to fit the ligand Remeber that box files are really molecules that are used to define the bounding box bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit boxes lig pdb gz If you want to add some padding to the boxes use the boxpadding flag For example to add 4 0 Angstroms to the box bin afitt e automate dir DATA grid grd ligand lig pdb gz protein nolig pdb gz exit boxes lig pdb gz boxpadding Finally
104. e to add new functionality or override certain default settings AFITT provides functionality for users to place their own startup files writtin in python for these purposes The startup file startup py should be placed into a user specific local directory on the computer currently running the application On Microsoft Windows 2000 and XP the startup file should be placed in C Documents and Settings USERNAME Application Data OpenEye AFITT 1 3 On Microsoft Windows Vista the startup file should be placed in C Documents and Settings USERNAME AppData Local OpenEye AFITT 1 3 On all other platforms the startup file should be in USERNAME OpenEye AFITT 1 3 14 3 Scripting Manual The complete details of the Python interface is fully described in a seperate API manual which documents every possible command that can be run within the application 14 4 Scripting Window As mentioned above Python is an interactive language and as such a scripting window is provided as an interface to Python The scripting window can be seen in Figure 14 1 Scripting Window ToolbarAdd ToolbarAddCombo Toolbar ddSeparator ToolbarAddSlider ToolbarVisibleGet ToolbarVisibleSet Figure 14 1 Scripting window with tab completion of Python commands The scripting window consists of two components input and output The large top component of the window is the output area which displays all of the output from commands entered in
105. e was created In addition the state file preserves the actual view layout and properties of that session State files provide an extremely convenient way to save sessions for later work or to share with collaborators Furthermore state files are the fastest method of reading and writing large data sets 30 Chapter 4 File I O Save File Save in O Temp ex Fa 4 4 My Recent Documents 3 My Network File name mystate oes Se Places Save as type DpenEye State Version 2 L 08S 92 a Cancel Figure 4 5 Saving state With the release of VIDA 3 0 a new state file version was introduced which is significantly smaller in size and faster to load than previous versions However this new version OpenEye State Version 2 cannot be read or written by VIDA 2 x To address that issue AFITT does provide the functionality to save OpenEye State Version 1 files which can be read and written by all versions of AFITT The new state version also supports a mini option which only writes out objects that are currently visible or referenced in the display bookmarks This can provide for an even greater savings in disk size and load time This mini state file can be further compressed by selecting the Compress grid contour information option in the General section of the application preferences Selecting this option zeros all non contour related positions in the grid which greatly improves its compressibility This sh
106. e with high refresh rates are becoming increasingly difficult to obtain For those machines that do not support hardware stereo split sreen stereo is still a viable option Both cross eyed and wall eyed views are available Furthermore the stereo angle and eye offset parameters can be adjusted in the preferences see Chapter 16 5 3 1 Microsoft Windows We have every expectation that most high end workstation class graphics cards e g 3DLabs Oxygen and Wildcat NVIDIA Quadro ATI FireGL will support hardware stereo but we have not been able to 44 Chapter 5 3D Display test all of them Furthermore no testing has been done on Vista and as such no guarantees can be made about stereo visualization on Vista One card that we did test which caused problems was the ATI FireGL V3200 There were no problems observed with the ATI FireGL V5000 5 3 2 Mac OS X Mac OS X has supported stereo in a window visualization since late 2005 and as such hardware stereo 1s possible on a Mac provided that the computer has an appropriate graphics card We have successfully test hardward stereo on a Mac Pro configured with the NVIDIA Quadro FX 4500 graphics card running both Mac OS X and Microsoft Windows via Boot Camp 9 3 3 UNIX Linux Currently the only UNIX platform that we know of that supports hardware stereo is SGI Irix We have successfully tested hardware stereo on a number of different SGI machines It is important to note that i
107. ecules match the pattern the user will be prompted as to whether to Mark the matching structures instead of selecting them as being selected would make them visible The third button inverts the current selection unselecting everything that was selected and selecting everything that is currently visible but was not previously selected The fourth button selects everything within a defined radius of the current selected set the default is 5 Angstroms The fifth button selects everything within a user specified radius of the current selected set This radius is specified by the slider in the bottom row of the pane The sixth button selects everything outside of the same user specified radius of the current selected set It is important to note that these distance based selection mechanisms expand their selected sets to include entire residues in the event that only a portion of a residue is included within the radius The last button contains a drop down menu which has different options based on the current selection If a molecule is selected it contains the following options Hide Outside Hide Inside and Restore Selecting one of these options will hide all of the atoms and bonds outside the current slider specified radius hide all of the atoms and bonds inside the current slider specified radius or unhide any previously hidden atoms and bonds respectively If a surface is the options are Crop Outside Crop Inside and
108. ed as command line arguments to the process in the order in which they appear in the main text entry box in the Process Editor see Figure 13 1 13 2 Pipes Process Manager Processes Status Omega Rocs Processes Pipe Rocs Pipe asa Omega Rocs Figure 13 6 Pipe Editor Once a number of processes have been created they can be piped together using the Pipe Editor see Figure 13 6 On the left hand side of the editor is a list of all the currently defined processes On the right is an ordered list of processes to be run and in which order to run top to bottom When chaining processes together into a pipe it is important that they have default input and output fields specified see Sections 13 1 1 and 13 1 2 The default output of a given process is passed into the default input of the following process When the final process is completed the user will be notified and the final output file will be handled based on its specification in the associated process CHAPTER FOURTEEN scripting AFITT is in part built on top of the Python scripting language which is an interactive interpreted strongly and dynamically typed language More information about Python can be obtained from the Python website located at http www python org The website contains many useful tutorials and other related information 14 1 Journal File All user interaction with the application goes through a Python layer even if the user is
109. ellow lines will appear in the control which indicate the position of the near and far clipping planes These can be moved by simply clicking on the triangular tab at the left of the lines and dragging them to the desired location The 3D display will update dynamically as these lines are moved In the center of the control is a small white dot that corresponds to the center of scene that the camera is pointing at The position of the center can be changed in the Z plane by dragging the dot in the vertical plane The position of the center can be changed in the X plane by dragging the dot in the horizontal plane Beneath this control is a slider which controls the scale of the text drawn in the 3D display The 3D display will update dynamically as the slider is adjusted 5 2 Rendering 43 Beneath the slider are three radio buttons which control the mode of stereoscopic visualization The default behavior is Off The Hardware option is only enabled on machines which are capable of performing 3D hardware stereo in a window This capability is determined by the computer s graphics card For more details on stereo display please see Section 5 3 5 2 Rendering The images shown in the 3D display are rendered using OpenGL therefore the quality and speed of the rendered scenes in the 3D display is dependent on the graphics card in the computer running the application Computers with graphics cards that do not support OpenGL acceleratio
110. eturn the absolute value of the float x floor x Return the floor of x as a float This is the largest integral value lt x fmod x y Return the floating point remainder of x y hypot x y Return the Euclidean distance y u y y Idexp x y Returns x x 2 log x base The logarithm of x to the given base If the base is not specified returns ln x log 10 x The base 10 logarithm of x pi The numeric constant PI or 3 1415926535897931 pow x y Return z radians x Converts angle x from degrees to radians sin x Return the sine of x measured in radians sinh x Return the hyperbolic sine of x sqrt x Return yz tan x Return the tangent of x measured in radians tanh x Return the hyperbolic tangent of x Table 11 1 Available math functions 11 5 1 Math Functions A large number of math functions are available for use in spreadsheet expressions as described in the previous section The currently available math functions can be found in Table 11 1 115 2 OEChem Functions A large number of molecular properties are available for use in spreadsheet expressions as described above The currently available molecular properties can be found in Table 11 2 11 6 Filtering 101 Name Property MW Molecular weight Num Atoms Number of atoms in the molecule Num Bonds Number of bonds in the molecule Carbon Hetero Ratio Ratio of carbons to hetero atoms in the molecule Returns 1 if there are no carbons in the m
111. ewness The skewness of the column e kurtosis The kurtosis of the column The statistics display is a dynamic view of the spreadsheet data and therefore when data is altered in the spreadsheet the statistics view is automatically updated For large data sets this may take some time and could cause editing the spreadsheet to be cumbersome To avoid this problem it is best to hide the statistics display until the editing process is complete 11 8 Formatting Columns There are a variety of different display styles for spreadsheet columns which can be edited on an indi vidual basis The customizable styles include color font significant digits and cell height The style for individual columns can be edited by selecting the Format Columns option in the top level Data menu This will launch the dialog seen in Figure 11 7 Column Format Column all Color By None Depiction Visible Minimum Locked E Marked Maximum 1 00 Compute VIDA Name i BRO Significant Digits 4 Cell Height 10 Figure 11 7 Spreadsheet format dialog On the left hand side of the dialog is a list of all the currently available spreadsheet columns including an All option Any formatting changes made in this dialog will be applied to the selected columns in this list On the right hand side of the dialog is a collection of formatting and display options At the top is a drop down menu of available coloring schemes which include e Red to B
112. f mol atom bond tri vert or surf This limits the matches for the part of the query it appears in to match either molecules atoms bonds triangles vertices or surfaces respectively Example type atom index This unsigned integer property limits the matches for the part of the query it appears in to items with the given index Each atom bond triangle etc has an index assigned when the item is created Since the indices are generally not exposed to the user this command is probably of limited utility without OEChem level access to the molecules in memory Example id 2 amp amp type atom amp amp index 10 15 5 Macros Pre defined sets 125 e query This string property performs a substructure search where the argument is treated as a SMARTS pattern and so limits the query to atoms which match the SMARTS pattern Example query cccn subset This string property matches all atoms which are in the previously defined subset with the given name Subsets may be defined via the Subset command so the subset query provides a way of creating shorthand references to other complex queries Example first de fine a subset Subset mysubset rn 5 amp amp id 2 Then the subset may be used as subset mysubset e key This unsigned integer property limits the match for the part of the query it appears in to match only objects with the given key Example key 100000001
113. f protein ribbons and c alpha traces respectively The next button makes all of the molecules in the current scope no longer hydrogen bond targets The last button in the row toggles whether or not external intermolecular hydrogen bonds are shown In the last row the first button allows the user to specify and turn on atom and bond labels The second button turns off all of the atom and bond labels in the current scope The third button toggles the dis 5 1 User Interaction 4 Contours Contours Radius Resolution a Normal View b Advanced View Figure 5 7 Contours pane of the Style Control play of non bonded atoms in the scene The remaining three buttons control the hydrogen style for the molecules in the current scope The first of the three turns all hydrogens on the second buttons shows only polar hydrogens and the last button hides all hydrogens Contours The contour pane of the Style Control is used to control the number and level of the individual grid contours as can be seen in Figure 5 7 a When the current application scope is set to Focused there will be a pulldown menu in the top left of the window containing a list of all the current grid contours If the scope is not set to Focused the scope will be displayed in place of the pulldown menu to remind users that all operations in this window apply to more than just the Focused grid Next to this area is a numeric display which displays the current contour
114. finements suma a ia Mee oe Ym HR E R Ge BE Be Hw MES Ligand Modeling Tel Ligand Modeling WindOW socs e abe be teh dae be eee hd el ae a 12 SD WMdOW s lt s nace eee bebe dee eee ed ew dae ea ee be ee heed LI Spreadsheet 4 24 4 8 ee GeO Oa em EWE ROR a Pee e TA OPENEN e oo a ald o ea a Be wk ta Se at i Gite Be iw hi ani a Locatins ligand density s casa a Se a RA e AE 7 6 Filtering Ligand Density Based on Distance to a residue 7 1 Working With Poor Density o lt s i320 4 22 a Pepe a eee es 7 8 Writing Refinement Dictionaries o 24 042 s s sr eva pee ew Ew ee es Protein Modeling Sell Bist Window oi e aeg ot aces Hoa Bh ha a SS Soe ga Ge ee ac Kare Maes G2 20 WINdOW a 2 25 84 abe a a He ole aog a ae a do ea So Style WINDOW ioi goe rae Gok we Hoe e ee wR ces ee PG we ew 84 Protein Modeling Operations 2 22 2 64 merece 8 5 Appending Prepending or Mutating Residues or Rotamers 8 6 Bond Speciiic Operavioms o eis sd a tes oe Sa ae da Qe ar de Bo Mouse Mode Toolbars i ones 4 acc one Gala ara se oa Ss a he a ee 8 9 2D Sequence VieW i 22 s arenos ae a moe be ed e ERE Ee oe ie BE ww aes 89 Ramachandran plot es eoma worre be ee ee EE ee a ee es 8 10 Modeling toolbar misas a Ae eR oe ee OE ee Ae aS Protein Refinement Ol Sethe Up Retmmacd ci s vad a Sh ow Bae ae oad Gear a Sd ws BOS we ew Sk 9 2 Altermg Refmac5 Parameters ooo ba Re Ee a ees 67 67 67 68 69 70 71 7
115. g a lasso style selection method by holding down the Right mouse button and moving the mouse in the window to define a selection rectangle a dotted rectangle will be displayed on the screen as you do this Releasing the Right mouse button will select all of the atoms and bonds inside of the rectangle The previous selection will be cleared unless the Shift or Ctrl key is also held down while moving the mouse Lasso style selection of surfaces and grids is not currently supported Additional methods of selection are available in the Selection pane of the Style Control see Section 5 1 2 Rotation The scene can be rotated in three dimensions by holding down the Left mouse button and moving the mouse in the window These movements will emulate rotation using a trackball The scene can also be rotated around just the Z axis by holding down the Alt key and the Left mouse button simultaneously Translation Using the mouse the scene can be translated in the XY plane by holding down the Shift key and the Left mouse button simultaneously Translation along the Z axis can be performed by holding down the Alt key Shift key and the Left mouse button simultaneously The scene can also be translated along the Z axis by holding down the A t key while using the mouse wheel Using the keyboard the scene can be translated horizontally in the display by pressing either the A key or D key which translate to the left and to the right respectively Scale
116. hange the other ET ET or Electrostatic Tanimoto grids are generated by OpenEye s Brood and EON applications When viewed in AFITT they behave and are treated essentially the same as Electrostatic grids see Section 5 10 1 FRED FRED grids are shape potential grids that are created by OpenEye s FRED application In AFITT the behave just like Generic grids see Section 5 10 1 except that their default contour color is purple instead of blue Generic 5 11 Surface Visualization 59 Generic grids are effectively the most basic grids and act as the primary grid workhorses They are completely customizable and have bare bones settings They default to having just one contour with a default color of blue Additional contours can be added as well as removed These default values can be changed in the Grids section of the application preferences 5 10 2 Reentrant Grids a Reentrant b Reentrant c Non reentrant Figure 5 18 Grid contouring examples showing a an infinite grid b the same grid rendered at a different center notice that the grid is rendered in the center of the scene not at the molecule and c a finite density grid showing the grid corners or extent of the grid Some grids have fixed extents and some grids are infinitely reentrant in space Reentrant grids obviously cannot be displayed in their entirety so instead only the region around the center of the current scene is displayed Grids that are being
117. he Alt key instead of the Ctrl key Additional methods of labeling atoms and bonds are available in the Style pane of the Style Control see Section 5 1 2 Menu A context sensitive popup menu can be generated by clicking in the window while holding the Right mouse button down This menu will contain operations that can be performed on those objects associated with the context when the popup menu was generated 5 1 2 Style Control The Style Control is a separate peripheral window see Section 3 5 2 which is provided to enable ad ditional interactions with the 3D display The Style Control is a vertical window consisting of many individual panes Each pane provides its own set of functionality The display of the individual panes can be toggled by clicking on the title bar of the pane of interest In addition individual panes can be torn off to act as top level windows by clicking on the icon in the top right of the title bar When 5 1 User Interaction 37 or amp AY Transparency J Figure 5 1 Color pane of the Style Control 6 99 the pane is torn off the icon becomes a icon which when clicked hides the torn off window The specific details of the individual panes are listed below Color AFITT provides a wide array of coloring options as can be seen in the color pane see Figure 5 1 The first button with the color wheel icon prompts the user to select a specific color which will be
118. he ligand and Vshape is the overlap between the ligand and the electron density A is a mixing parameter that represents the degree to which the shape of the density to dominate the combined potential 10 This function is optimized while the strain placed on the ligand is bounded producing high quality fits with low strain ligand conformations For each ligand there are columns in the Results spreadsheet that should be noted 1 Tanimoto Shape This is the measure of how much the original conformation matched density 2 Tanimoto MMFE Shape This is a measure of how closely the final result of the combined force field matches the shape of the density Note that this will never be an exact match 3 Local Strain This indicates how much strain was added to the original conformation during the fitting process 4 Lambda This is the scalar indicating which component of the combined forcefield was used gt 1 implies that more precedance was given to the Shape force 1 e density lt 1 implies that more weight was given to the MMFF force In general the higher the Tanimoto MMFF Shape value the better the fit to density There is one impor tant caveat Tanimoto MMFF Shape can only be used to compare ligands being fit to the same blob They are not comparable across different density regions However when AFITT fits multiple stereovariants to the same blob sorting the Tanimoto MMFF Shape column will percolate the highest scoring ligand
119. his script as documented in the file do_afitt_gold sh bat This is an example shell script that shows the command line that can be used to fit the ligands in the supplied gold data set The bat version is the appropriate version for Windows This script is located in the supplied data directory CHAPTER NINETEEN Automation AFITT was not desigened to be an automation system but it can serve this purpose OpenEye suggests that for ligand fitting the flynn command line tools be used instead of AFITT AFITT s automation tools are slowly being deprecated in favor of command line automation That being said Automation in afitt can be performed in several ways 1 Scripting an example python test script is given in the examples directory This is a very powerful script that can be used to customize automation 2 Built in automation AFITT has a method of performing automation from the command line as described below The major downside of this automation is that it is not entirely command line based The AFITT window appears along with the various graphics window While it is nice to see the process unfold this does take consume the computer s resources and is quite a bit slower than using the command line tools In addition to scripting AFITT includes powerful automation from the command line This automa tion includes the ability to search arbitrary directories on disk and find ligands proteins and grids and automatically
120. iated file There is an Open All option at the bottom of the recents menu which will load all of the files listed in the recents submenu Files can also be loaded by specifying them on the command line when starting AFITT or by dragging the file of interest onto the AFITT desktop icon By default there can only be one instance of AFITT running at a time so if an instance of AFITT is running and a new file is specified on the command line or if a file is dragged onto the desktop icon that file will be loaded in the currently running instance instead of loading a new instance If desired multiple instances can be enabled by selecting the Allow multiple instances option in the application preferences see Chapter 16 In addition to the Open menu item there is a separate Open State item which filters out all files except AFITT state files State files can be opened using any of the mechanisms described above but their special nature warranted their own menu item For more details on state files see Section 4 1 6 There is also an Open Special submenu which contains a Script option which like the Open State option filters out all files except for Python scripts The Open Special submenu also serves as an excellent target area for adding custom open operations via the scripting interface see Chapter 14 4 1 1 Molecules Multiple molecular file formats are supported for reading and include e OpenEye
121. iated list in the List Window see Chapter 12 and select the Delete option This will close the file and remove the contents from the application However if an object is present in multiple lists deleting one of those lists will not delete that object from the other lists It is important to note that deleting a list does not affect the actual file that was read To close all of the currently loaded files select the Clear All option in the File menu In addition to clearing everything that is loaded this option effectively restores the state of the application to what it was in when the application was started 4 3 Saving Files AFITT is capable of saving multiple types of files including molecules grids surfaces and state files The details of the individual formats can be found in the relevant sections below A file can be saved by choosing the Save option the File menu This will launch a dialog prompting the user to select which objects to save see Figure 4 4 There are two sets of buttons at the top of this dialog which provide mechanisms for filtering the available options The three buttons on the top left toggle the display of molecules surfaces and grids respectively The two buttons on the top right provides filters for showing only the Marked and or Visible objects respectively Once one or more objects have been selected and the OK button is pressed a file dialog will appear allowing the user to specify
122. ich the application is installed Nothing else is required 2 3 Mac OSX The Mac OS X version comes pre bundled All that is required is to drag the AFITT icon to the desired location on the computer such as the Applications folder Double clicking on the icon will launch AFITT The first time AFITT is run AFITT may prompt for the location of the license file If this occurs there will be two AFITT icons in the Dock until AFITT is restarted To uninstall AFITT simply drag the AFITT icon to the trash from the folder in which it was installed Nothing else is required When AFITT is run it obtains all of its environment variables from the users profile file Users of shells besides the bash shell must still alter the profile file in order to pass environment variables through to AFITT 2 3 1 Platform differences Microsoft Windows and Linux frequently use Ctrl keyboard combinations For all menu operations such as copy paste and undo Mac OS X uses the Apple Command button instead Similarly to display information on mouse over in the 3D window the Alt Option button must be held down instead of the Ctrl button 2 3 2 Launching from the command line In a terminal window AFITT can be launched from the command line as follows open a afitt as long as AFITT is in the search path If AFITT is installed into the Applications directory it will automatically be in the search path 2 4 Licenses In order to run AFITT a vali
123. ied to all surfaces and grid contours in the current scope How ever if a part of a surface is selected the transparency will only be applied to the selected region as can be seen in Figure 5 2 b Selection The simplest mechanism to perform a selection is to click on the object of interest in the 3D display using the mouse as described in Section 5 11 3 However a number of addition selection mechanisms are available in the selection pane as can be seen in Figure 5 3 In the top row of the selection pane there are seven individual buttons The first button with the A icon selects all of the currently visible molecules The second button with the partially selected molecule icon performs a selection based on a substructure query Clicking on this button will launch a query dialog which allows the user to specify a substructure query The substructure can be entered as a SMARTS 38 Chapter 5 3D Display a Transparent surface b Partially transparent surface Figure 5 2 Transparent surfaces with underlying ribbon display visible Selection AMSARAAS O Figure 5 3 Selection pane of the Style Control pattern selected from a large number of predefined patterns or can be specified using a common or IUPAC name which will be converted to a structure using OpenEye s Lexichem toolkit All atoms in the current scope matching the specified pattern will be selected If the current scope is set to All and more than 50 mol
124. ight clicking on a reflection data set in the list widget shows the Maps menu option while right clicking on a molecule adds the Set crystal params option which allows the user to set or modify the unit cell and symmetry information on a molecule 70 Chapter 6 Modeling Basics 6 5 Real Soace Refinement AFITT fits ligands and proteins to density in real space For ligands this usually means that some portion of a model exists to phase the density in order to reveal the density of the ligand AFITT supplies some useful facilities to assists in preparing complexes for real space refinement 6 5 1 Density Versus Blobs One of the issues that may be encountered is the difference between density and blobs A blob is not density rather it is a density boundary Blobs are used to bound the density to use during various proce dures In AFITT 3D surfaces are used to indicate these regions This allows all surface operations such as pruning and carving to be applied to blobs For example if the blob is too large it can be cropped to remove the undesired regions For more details on operating on surface please see Chapter 5 11 6 5 2 Best Practices Based on analyzing several hundred protein ligand combinations the best way to use AFITT for ligand fitting is described as follows of course your milage may vary e Always use a protein The protein is used to identify modeled density where the ligand cannot be placed Without this
125. igned to intuitively and effortlessly handle very large data sets while still generating extremely high quality images It provides multiple modes of display including 1D 2D and 3D Furthermore the VIDA Framework contains an excellent interface for data analysis as it includes a chemically aware spreadsheet and a powerful list based architecture AFITT includes capabilities from both OpenEye s Shape and MMFF toolkits as well as powerful new functionality of its own to enable model building shape fitting map calculation and display and real space refinement using shape and MMFF AFITT also provides interactive Ramachandran plots and an interface to external refinement programs This manual is divided into several chapters Installation should be read by users and administrators that need to install the software on various platforms The Changes chapter describes the changes since the last version Otherwise the Getting Started chapter provides a good overview of AFITT and it s capabilities and can be used as a quick start for the anxious crystallographer The Ligand Modeling and Manual Modeling sections in the Modeling chapter go into more depth on the techniques used to fit small molecules and proteins into density The final chapters describes detailed usage of the various widgets available to AFITT CHAPTER TWO Installation This chapter provides the necessary details required to install AFITT 1 3 Currently supported platforms in
126. ikelihood method Acta Cryst D Vol 53 pp 240 255 1997 Brnger A T Adams P D Clore G M DeLano W L Gros P Grosse Kunstleve R W Jiang J S Kuszewski J Nilges M Pannu N S Read R J Rice L M Simonson T and Warren G L CNS CNX Crystallography amp NMR System A New Software Suite for Macromolecular Structure Determination Acta Cryst 1998 D54 905 921 Lovell S C Word J M Richardson J S and Richardson D C The Penultimate Rotamer Li brary Proteins Structure Function and Genetics Vol 40 pp 389 408 2000 McGann M R Almond H R Nicholls A Grant J A Brown F K Gaussian Docking Func tions Biopolymers Vol 68 pp 76 90 2003 Bower M J Cohen F E Dunbrack R L Jr Prediction of protein side chain rotamers from a backbone dependent rotamer library a new homology modeling tool J Mol Biol Vol 267 pp 1268 1282 1997 Robert J Fletterick Raymond Matela Color coded a carbon models of proteins Biopolymers Vol 21 pp 999 1003 1982 Matthew D Eldridge Christopher W Murray Timothy R Auton Gaia V Paolini and Roger P Mee Empirical scoring functions I The development of a fast empirical scoring function to esti mate the binding affinity of ligands in receptor complexes Journal of Computer Aided Molecular Design Vol 11 pp 425 445 1997 Araz Jakalian Bruce L Bush David B Jack and Christopher I Bayly Fast Efficient Genera
127. image of the resulting layout The Save option allows for the creation and storage of custom layouts The Organize option enables the reorganization of the ordering of the layouts 3 5 1 Main Windows The main window is the window that occupies the central area of the application on the screen At any given time there can be only one main window however there are multiple windows which are capable of being the main window if desired The choice of main window can be controlled by selecting the desired option from the Main window submenu in the top level Windows menu In Figure 3 2 the 3D display see Chapter 5 is serving as the main window Currently there are three different main window options the 3D display the 2D display see Chapter 10 and the spreadsheet see Chapter 11 More details about the individual windows can be found in their relevant chapters 10 Chapter 3 Getting Started Focused Visible Marked List Window Name 1A28_prot mtz gt 1A28_input pdb 1A28 pdb ligand 1A26_afitt_lig pdb Modeling 1A28 pdb ligand PR 10 10 99 999 9 JEJE Regular Map 2Fwt 1428_prot mtz Difference Map None gt y al al Lo K z Color esr oseo Transparency mana OLI PELINLIMS TE PIVIYACHONTKPDTSSSLLTSINOLCEROLLSVVKWSKSLPCERNLHIDDOT TLI QY SUMSIMVEQICNRSYKHVSCCMLYFAPDLTINEQRMKESSE OLB EEI KVLLLINTI PLEGLRSQTOFEEMRSSYIRELIKATGLROKGVVSSSORFYO
128. ing Files s gsr aoo e hog dew a a eR da de oe es 27 4 3 Saving Piles cocina a a eee A A R A A A A G 27 44 Importine Files 20 45 2484 208 bdo PEM aE Ae Rae e a a 30 4 5 Exporting Files o siea Garo ba ERAS ALR W a e ae eai Ra A Sa AA i 31 46 Drac and Drop ci sooi a dia aaa RR Oh ae ee Pe a ee 32 3D Display 34 Hi Ween interaction il a amp Ave ae ee eer ee howe ds ooh aed Ghee dg Bate de DE 3 2 Rendering a c goi poat sare hw Mee we Od ESO Re e ERTS POR a ia BB wer eS DI PITO ig ee Eee e Pate PVE Da ee eR ORS EE ee berets Dia Viewpoints sasae ea heck ow ROE WD he eke We Pe RG we Ak RA A S BOOKMARKS ooe a 242 cRNA Ea ER ER a A a EAR EER EE eo 5 6 Tiled Display aoine hock And Bw a A ke A RS AAA BS Det MOOR ANS sa cute qe ge atest eck ee Ge Scere goog gh Bled a ee ee Gra ae cee 3 9 Display Widgets ne vo ee kde a a eee a ee ad ew ea eR 5 9 Molecular Visuali zation ect Goce Bee a Hd ded Ra a oe we 3 10 Grid Visualization csc ee a ee eh a Bd ee tw ed 5 11 Surface Visualization s oe e 0 8 teera ee ER ERE ee 312 Symmetry onc ik ee yaaa e RD ERE DME ER EY e eS OS 513 Symmety MEDI e a ee Sati SoM Sak Gok Ym SR E R Ge Bae a Mw MES Modeling Basics Gil Tasks sai seid ds oh eed a te ee a he heed we Ad eb ed Re ele cg Gol ete 02 FHGN cuco mara a Oe ooh eed eae os Bah ee yw a E 63 EditmenW c 2 acra e Seb ee le ee ate betwee ended tee a Sees 64 Popup Mens o sas s wewa eeg Reem ER EERE HEME OH REO eS 63 Real Space Re
129. is currently focused When deaeling with small moleculees this view may be unecessary as the current ligand s 2D structure is also visible in the 3D Window 3 10 How to Center 13 3 10 How to Center The default method of centering is by focusing or selecting a molecule and then clicking the Center button in the upper toolbar This button is next to the Screenshot button that looks like a camera If an atom or set of atoms are selected the Center button will center on the seleted atoms Alternatively right clicking on a molecule in the 3D Window List Window or spreadsheet will popup a menu that includes an option to center Selecting center from this menu will center on the object 3 11 Tasks AFITT contains several workflows to assist in various aspects of protein and ligand modeling These workflows are called Tasks and can be selected from the Task menu or from the Layout widget in the upper toolbar Each Task has a default layout designed for the respective workflow If the layout is changed AFITT remembers the changes allowing the user to tailor the workflow to suit their needs The basic tasks are Ligand and Manual The Ligand task focuses the workflow on real space refinement of small molecules in the corresponding density The Manual task uses OpenEye s shape fitting and molecular forcefield techniques to assist the manual fitting of proteins to density Each workflow is described in detail in Chapter 6 however the basic
130. ise all residues are shown Clicking on a residue from the plot will select the residue for operations Additionally hovering over a residue will show the residue information The Richardson Ramachandran plots are to some degree specific for given amino acids e g glycine proline pre proline For this reason glycine proline and residues immediately N terminal to proline don t show up in the Richardson general Ramachandran plot only in their respective plots 8 10 Modeling toolbar The modeling toolbar shown in Figure 8 8 is a simplified version of the protein modeling widget Append or replace Residue Append or replace nucleotide SP ARNDCQEGHILKMFPSTWYVCAGUT Figure 8 8 Ramachandran Plot If a residue is selected clicking on an amino acid code in the toolbar will mutate the residue If no residue is selected the amino acid will be appended to the end of the protein The toolbar uses the secondary structure used in the protein modeling controls see Figure 3 9 CHAPTER NINE Protein Refinement Refmac refinements can be launched by using the Task Launch Refinement menu option By default the visible molecules and reflection data is used to launch the refinement The refinement dialog is seen in Figure 9 1 9 1 Setting Up Refmacs For external refinements to work refmac5 must be installed and available from the command line The Destination field contains the directory and initial name for the refine
131. isk space than an entire grid Graphics The graphics control pane in the Style Control provides an additional level of control over and interaction with the the 3D display see Figure 5 8 It is particularly useful for users without mouse wheels 42 Chapter 5 3D Display Depthcue Slabbing Mirror Text Scale Stereo off Splitscreen O Hardware Figure 5 8 Graphics pane of the Style Control At the top of the graphics pane are three check boxes Depthcue Slabbing and Mirror The first two check boxes toggle the use of their associated properties respectively The Mirror check box is only enabled when slabbing is on and controls whether the front and back slabbing planes move together mirrored around the center of the scene or independently Beneath these check boxes is an interactive control which allows the user easy control over both depthcueing and the clipping planes Displayed in the control is a top down view of the current scene in the 3D display The front of the scene is at the bottom as indicated by the label When depthcueing is enabled two red lines will appear in the control which indicate the start and the end points in the depthcueing calculation These lines can be moved by clicking on the triangular tab at the right of the lines and dragging them to the desired location The 3D display will update dynamically as these lines are moved When clipping or slabbing is enabled two y
132. it might belong to When the last object is removed from a list the list itself will be removed 12 6 List Toolbar This List Window installs a collection of five navigations button in the application toolbar which can be see in Figure 12 5 These buttons provide an additional mechanism to browse through lists and their contents in AFITT In addition these buttons are aware of the current list view as described in Section 12 3 and as such operate on that view The first four buttons in the set browse to the beginning to the previous object to the next object and to the last object respectively The final button with the play icon initiates a slideshow which steps forward to the next object after a certain period of time this can be set in the application preferences The slideshow can be paused by clicking on the same button again which should have changed to have a pause icon once the slideshow started The slideshow will automatically stop when it reaches the end of the objects in the current view of the List Window CHAPTER THIRTEEN Process Management The Process Manager allows the user to start monitor terminate and load results from processes external to AFITT As part of this functionality it is possible to write out currently loaded molecules or grids to files which can be used as input to the external processes Furthermore it is possible to create pipes which chain together the input and output of indivi
133. ity O White on Black O Black on White E show Bond Order Show Dipolar Bonds 30 Display Hydrogens Oa Polar O None Bond Ball Ratio Atom Colors Line Width pixels O Dark Background Scheme Residue Radius A Auto Apply on Background Color Change Color Restore Apply _ save _ cancel Figure 16 2 Molecule Display Preferences 16 1 Useful Preferences 131 16 1 2 Changing Mouse Map The default mouse map is shown in Chapter 20 Additional mouse maps are available from popular crystallography or visualization programs The default mouse map can be changed in the Molecules preference The available mousemaps are e AFITT e COOT e Insight e Maestro e MOE e 0 e Quanta e PyMol e RasMol e Sybyl e VIDA Preferences General General OpenGL Depictions Molecules Grids Display Colors Surfaces Antialias Lines Auto Center Panes Annotation Background 7 Yellow Modeling Ligand Clipping Slabbing C Auto Center Annotation Foreground Il Black Depthcue Auto Fit Labels White D Tied C Show Axes Marked F fed292 Fixed Size Labels C show symmetry Reference E Green Use Nice Fonts Show Titles Selection i 78000 Show Toolbar Indicators Titles White Render Quality Screenshots Show Active Border Show Tile Borders MS Shell Dig 2 Default Width pixels 400 MS Shell Dig 2 Default Height pixels 200 Interaction Matrix Mode Placement Mouse Map O With Pa
134. level Next to this display are two buttons one with a icon and one with a icon These two buttons allow for the creation and deletion of individual grid contours Please note that these options are disabled for both Electrostatic and ET grids as they can have only two contours positive and negative Furthermore the contour levels of the positive and negative contours are coupled together and so changing one will change the other but with the opposite sign At the bottom of this window is a slider which allows for direct control over the contour level To the right of the slider is an Advanced button with the equalizer icon which will expand the pane to show a number of advanced options see Figure 5 7 b In the advanced section there are two adjustable parameters Radius and Resolution and one button The parameters control how contours of Reentrant grids are displayed according to their symmetry The Radius parameter specifies how far out from the center of the scene in Angstroms AFITT should look in the grid in order to generate of the isocontour The Resolution parameter specifies the resolution in Angstroms of the sub grid used to generate the isocontours The single button with the check icon to the right of the Radius parameter converts the current grid contour into a fixed surface independent of the grid This can be particularly useful when generating state files as a single surface can take up considerably less d
135. loaded into a new list called Pasted The ability to load molecules from other applications depends on how the other applications place molecules on the clipboard Cur rently supported clipboard formats include SMILES ChemDraw CDX ISIS Sketch MDL SD Files and OpenEye OEB files Furthermore for other molecular visualization programs that support a drag and drop interface molecules can be dragged from the List Window into those applications e Dragging molecules from the List Window into other applications will cause molecules molecular data or molecular images to be transfered to that application depending on how that application expects to receive data For instance dragging molecules into a Text Editor will result in a list of SMILES corresponding to the dragged molecules to be entered into that application e Dragging a 2D depiction from the Spreadsheet see Chapter 11 will cause that image to be loaded in the associated application if possible Along the same vein a similar copy and paste interface exists for passing molecules images of molecules and molecular data between applications The Copy option can be found in the Edit menu and can also be performed by pressing Ctrl C on the keyboard This will copy all of the molecules in the current scope to the clipboard unless the active window is the Spreadsheet see Chapter 11 in which case it will copy all of the selected Spreadsheet data to the clipboard instead The P
136. loating point property matches atoms with the the given formal charge Example Q gt 0 radius Radius This floating point property matches atoms with the the given radius Example radius lt 1 4 ac Atom color This unsigned integer property matches atoms with the the given atom color The color should be specified as a packed integer e g from OEggColor GetPackedColor Example ac Pink GetPackedColor elem Element number This unsigned integer property matches atoms with the given element number Example elem 6 weight Molecular weight This floating point property matches molecules with the given molec ular weight Example weight gt 10000 IsAminoAcid This boolean property matches atoms whose residue names is the same as an amino acid residue recognized by OEChem Example IsAminoAcid 1 Equivalent to r aa IsNucleicAcid This boolean property matches atoms whose residue names is the same as a nucleic acid recognized by OEChem A C G T U Example IsNucleicAcid 1 Equivalent to r dna IsWater This boolean property matches atoms which are in a water molecule as recognized by OEChem Example IsWater 1 Equivalent to r Swat IsSubstrate This boolean property matches atoms which are not protein nucleic acid or wa ter as determined by the above queries IsSubstrate 1 Equivalent to IsAminoAcid 0 amp amp IsNucleicAcid 0 amp amp IsWater 0 99 66 1 type This string property may be one o
137. lob Picking tool in the modeling widget individual density regions can be selected from the 3D window Simply click on the Blob Picking button and then right click on a piece of density in the 3D window 4 SCiNi Generates a shape contour of the active sites The Generate Negative Image option of the Task menu attempts to find the active sites of the protein This generates a Shape Contour Negative Image by searching the protein space with a collection of small molecule fragments The resulting shape potential can be used to as a type of density from which to manually pick blobs This process may take some time so be prepared to go out to lunch 3 13 Protein Task The Protein Mode gives complete control over atom positions in density The initial view of the manual mode is seen in Figure 3 7 While the initial view of Protein Mode may initially be intimidating there are only a small handful of operations that are used regularly These operations also have hot keys and key stroke combinations to facilitate usage for advanced users For complete details see Chapter 6 To perform modeling operations one must select the desired structure or residue prior to performing an operation During some modeling operations such as rotating and W angles the behavior of the mouse will alter to perform the requested operation The current operating mode is indicated at the top right of the 3D Window In order to revert to the normal mod
138. lue e Blue to Red 104 Chapter 11 Spreadsheet e Rainbow e Rainbow Backwards e Red Yellow Blue e Grey to Green The selected coloring scheme will be applied to the column using the scale specified beneath the drop down menu determined by the minimum and maximum values The default values are 0 0 and 1 0 respectively but the current minimum and maximum values for the selected column can be computed by clicking on the Compute button to the right of the minimum and maximum fields 11 9 Importing Exporting 11 9 1 Importing Spreadsheet data can be imported from external files in a variety of formats including comma separated values csv and tab delimited values txt To import a file select the Spreadsheet option in the Import submenu of the top level File menu This will launch a dialog to assist in the import process which can be seen in Figure 11 8 Spread Sheet Import Options Z Combine repeating delimiters stip First V First row is header 0 Ignore Empty Headers 0 Click on a header to change column name Family 1CS0_uM set 0 9600 0 9500 0 9400 0 9200 Figure 11 8 Spreadsheet import dialog At the top of the dialog are a number of options to specify the format of the file to be imported and at the bottom is a quick preview of what AFITT thinks the first five rows are The preview will update dynamically to match any change
139. m E E cs E E El El EE EA AE A AAA EE Figure 12 3 Child object mimicing parent useful to identify specific objects when running various commands Each top level object has its own unique ID but child objects like conformers and atoms are all a part of their parent molecules s ID Columns can be shown or hidden as desired by right clicking in the header of the List Window These changes will be remembered in the Preferences see Chapter 16 and thus persist between sessions The Visible Locked and Marked properties can be toggled by clicking on their associated columns in the List Window If multiple objects are highlighted simultaneously and the click occurs in a column associated with the highlighted region that change will apply to all of the highlighted objects Compare Figure 12 4 to Figure 12 1 In Figure 12 4 the highlighted objects have been made Visible by clicking in that column The Focused object has changed as a result as well 12 5 List Manipulation The List Window operates on the basic concept that every potentially viewable object is placed inside a list As a result the root items in the List Window are always lists These lists can be combined subsetted and manipulated into new lists The properties of all the objects in a list can be set together by clicking in the columns of their containing list Finally objects can easily be removed from and inserted into lists 12 5 List Manipulation 111
140. matically cleaning up a protein AFITT includes a Mark State and Revert to last mark facility to quickly make a checkpoint of the current session and restore it quickly seen in Figure 8 2 The breakdown of the modeling widget is as follows 8 4 Protein Modeling Operations These commands attempt to automatically fit the modeled atoms to density using OpenEye s various shape and forcefield tools MMFF Shape Fit Selection Shape and Torsion Fit Mark State Regularize Selection Revert to last mark E 53 63 6 E EX al 0 n Up Protein Rigid Shape Fit Selection Pep Flip Find Best Rotamer Figure 8 2 Protein Controls e MMFF Shape Fit Shortcut SHIFT G This takes the current conformation and uses the MMFF force field plus a shape force to fit the selected atoms to the visible density e Torsion Shape Fit Shortcut SHIFT D Uses torsion driving plus the Shape force to drive the residue into density When this works it works well though in some cases it generates non physical structures A combination of torsion driving and then MMFF Shape Fit seems to work well e Regularize Shortcut g This corrects poor chemistry and bond angles between the selected atoms It is a pure geometric energy minimization and any available density is ignored 8 4 Protein Modeling Operations 83 Best Rotamer Search Shortcut 0 This searches the Dunbrack or Richardson rotamer libraries to find the highest probability rotamer for
141. ment job If the path is My Documents afitt_refine then launching the refinement will produce several output files e My Documents afitt_refine sh the shell script that can be used to relaunch the refinement job on unix e My Documents afitt_refine bat the batch script that can be used to relaunch the refinement job on windows e My Documents afitt_refine param the refmac5 parameter file used for the refinement Destination Path Refinement Type normal or rigid Molecules To Refine Refinement cycles Figure 9 1 Refinement page of the Modeling Window 89 90 Chapter 9 Protein Refinement e My Documents afitt_refine pdb the input pdb file used for the refinement automatically gen erated by AFITT e My Documents afitt_refine cif the input refinement dictionary used for the refinement au tomatically generated by AFITT Method can be either Normal or Rigid which speficy a flexible or rigid refinement respectively When the Refmac Launcher first appears the Molecules list is populated with the currently visible molecules If these are not desired the appropriate molecules can be selected from by clicking on the button to the right of the molecules list The Reflection Data field allows specification of which reflection object currently this should be a mtz file should be passed to the refinement Note that AFITT does not write reflection data but merely
142. mple to append a glycine residue to the end of the current protein simply click on the G button in the residue toolbar as seen in Figure 3 13 Append or replace Residue Append or replace nucleotide SP ARNDCQEGHILKMFPSTWYVCAGUT Figure 3 13 Residue Toolbar e Ramachandran Plot The ramachandran plot shows the and W status of the protein resdiues Clicking on a residue in the plot will select it in the 3D Window Once a residue is selected the Mouse Toolbar can be used to manipulate the Y angles e 2D Sequence View The 2D sequence view displays the protein sequence of the currently visible molecule If a ligand is Focused then it shows the 2D structure of the ligand Residues can be selected by clicking on the appropriate sequence Right clicking on a selected residue will displays a popup menu that includes various modeling operations that can be used as short cuts 22 Chapter 3 Getting Started 3 14 Refinement AFITT automatically prepares protein and ligand input for use with CCP4 s refmacS protein refinement tool and X PLOR CNX AFITT generates refinement dictionaries using the MMFF94 force field including bond lengths periods and chiral volumes AFITT also identifies and includes proper LINK records for covalent bonds refmac5 can be launched from the Task menu by selecting the Launch Refinement option As long as refmac5 can be run from the command line AFITT will be able to launch and refine proteins
143. n order to properly view stereo on an SGI the screen resolution must be set to a value that is appended with the letter s For instance 1200x1024 would likely not behave correctly whereas 1200x1024s would 5 4 Viewpoint When viewing objects in the 3D display there is always a center to the scene where the virtual cam era is pointing When the first molecule grid or surface is loaded into the application the center of that object becomes the center of the scene where the camera is pointing By default the center does not automatically change with changes to the scene The center can be changed explicitly from the main application toolbar the right click popup menu or using the following scripting commands ViewerCenterSet or ViewerCenterAndRadiusSet Furthermore the center can also be trans lated using the mouse interactions described in Section 5 1 1 The center can be made to automatically change when browsing if the Auto Center or Auto Fit preferences are set to true the default is false The Auto Center preference directs the window to always recenter the scene after any changes are made to that scene The Auto Fit preference directs the window to not only recenter the scene after any changes are made but to also ensure that everything in the scene is comletely visible in the window These preferences can be edited in the applications preferences dialog see Chapter 16 5 5 Bookmarks 45 5
144. n either the 3D see Chapter 5 or 2D see Chapter 10 display Selected objects are usually indicated by a change of color the default is orange in these displays More details on the actual process of selection can be found in the 3D and 2D display chapters 8 Chapter 3 Getting Started 3 4 Scope Scope is not a specific state or property like those discussed in the previous section but is rather an indicator as to which objects the application should operate on The default application scope is Focused which means that all of the application functions will operate on the Focused object see Section 3 3 1 Selected Figure 3 1 Available Scopes However as seen in figure 3 1 if there is anything currently Selected see Section 3 3 5 the selected set will take precedence over the current scope Furthermore in Focused scope if there is no Selected set and no Focused object the Focused scope defaults to behaving as if it were the Visible scope The Visible scope operates on all objects that are currently Visible see Section 3 3 2 Locked see Section 3 3 3 and or Focused The Marked scope operates on all objects that are currently Marked see Section 3 3 4 The All scope operates on every object currently loaded in the application Operating on the Al scope can be a very lengthy process and as such its progress is displayed in the progress bar in lower right hand corner of the application Fortunately operations in the
145. n will still be able to display images but the quality and speed with which the images are displayed may be markedly reduced To help alleviate situations like this multiple levels of detail are supported ranging from Fastest to Best which adjust the quality of the rendered scenes accordingly For most computers purchased within the past year Best level of detail should be appropriate In addition to the level of detail the scene can be further adjusted by modifying a few select OpenGL parameters such as the background color as well as the lighting and material models All of these param eters can be adjusted in the application preferences see Chapter 16 5 3 Stereo Both hardware and split screen stereo displays are supported in the 3D display Hardware stereo requires a graphics card that supports stereo in a window display as well as the appropriate stereo glasses The current version of hardware stereo has been successfully tested on Microsoft Windows Mac OS X and SGI Irix using CrystalEyes glasses from StereoGraphics In addition to the stereo capable glasses a CRT monitor with a high enough refresh rate is required We recommend a refresh rate of at least 100 Hz with 120 Hz being preferred as the effective refresh rate of the monitor is halved due to the fact that the monitor has to swap two different scenes back and forth to create the stereo effect Unfortunately CRT monitors in particular thos
146. nEye This product should not be used in the planning construction maintenance operation or use of any nuclear facility nor the flight navigation or communication of aircraft or ground support equipment OpenEye Scientific software shall not be liable in whole or in part for any claims arising from such use including death bankruptcy or outbreak of war Windows is a registered trademark of Microsoft Corporation Apple and Macintosh are registered trade marks of Apple Computer Inc AIX and IBM are registered trademarks of International Business Ma chines Corporation UNIX is a registered trademark of the Open Group RedHat is a registered trade mark of RedHat Inc Linux is a registered trademark of Linus Torvalds Alpha is a trademark of Digital Equipment Corporation SPARC is a registered trademark of SPARC International Inc SYBYL is a registered trademark of TRIPOS Inc MDL is a registered trademark and ISIS is a trademark of MDL Information Systems Inc SMILES SMARTS and SMIRKS may be trademarks of Daylight Chemical Information Systems Macromodel is a trademark of Schr dinger Inc Schr dinger Inc may be a wholly owned subsidiary of the Columbia University New York Python is a trademark of the Python Software Foundation Java is a trademark or registered trademark of Sun Microsystems Inc in the U S and other countries The forefront of chemoinformatics is a trademark of Daylight Chemical Information Systems Inc
147. ne Window cirio eke bee Ew Oe eR ae A ae a 145 OBChembiie 4 dea ed a ae eed A eR ae AS ees Query language 15 Antrodtiction dedo ice Be Gow ar woh e Dede Ged we Bog ew aa 15 2 Operators ee c g edhe wy RO oh ee ee ERE OES Ee aoe ee 133 Lists and Ranges kaa kh a eee A OER EAA eS OS 154 Properes ich ade a Ma ae ORS Cakes bee he he eae A E 15 5 Macros Pre defined sets isc ie ee a ae ee ee ee es 15 6 Scripune with SeratehSeope wa swt a ee eG a a Pa Be eR rs dG Preferences 116 1 Usel Preferences ce 0 ho hk e e Ge ER Rea ee Oe PRR Ge a Example Data Example Scripts 107 107 107 108 108 110 112 113 114 117 118 118 118 119 119 120 122 122 122 123 123 125 126 128 130 133 134 19 Automation 19 1 Getting Started With Automation sses csta spiew aa woa R0 esaa 19 2 Advanced Automation s aeu o aia oa a i ea Oe AAR Sw ee 19 3 Plato DUNES e a ir BR Ge A A E a NA r ie Ae AUER Se ee 20 Mouse Maps QUA ARTT Mouse Naps acc a aui aan me Bee a 20 Era a a a RE Ao a ae 21 Release Notes 21 1 Version 1 3 1 21 2 Version 1 3 0 21 3 Version 1 2 2 21 4 Version 1 2 1 21 5 Version 1 2 0 Bibliography 135 135 136 140 141 141 142 142 143 145 147 149 151 CHAPTER ONE Introduction AFITT is a crystallographic model building program built on top of the VIDA Visualization Frame work which provides the core of OpenEye s main visualization application The VIDA Framework is des
148. nges In addition to being able to specify a single value for a property it is also possible to specify a list of values or a range of values Lists and ranges are provided as a convenience since they allow queries matching groups of atoms in a compact manner e LIST A list is a series of values enclosed in square brackets for example rn 4 12 38 This is exactly equivalent to rn 4 rn 12 rn 38 each listed residue is selected It is also possible to negate a list rn 4 12 38 selects all residue except 4 12 and 38 and is therefore exactly equivalent to rn 4 amp amp rn 12 amp amp rn 38 e RANGE A range is a pair of values enclosed in parentheses e g rn 1 10 All residues between 1 and 10 inclusive are selected so this command is exactly equivalent to rn gt 1 amp amp rn lt 10 Negated ranges are also possible rn 1 10 is equivalent to rn lt 1 rn gt 10 and therefore selects atoms not in the given range Lists and ranges are only valid with the operator and the operator It is not legal and nonsensical to try to evalate an expression such as rn gt 1 10 15 4 Properties The following properties are defined and may be used for selection e id This unsigned integer property limits the match for the part of the query it appears in to match only object with the given ID as shown in the list window Example id 3 e a Atom name This string property matches the atom name Example a CA
149. not aware of it For instance every button in the application is actually associated with a Python command that is sent to the internal interpreter when that button is pressed The same integration is true of all the menu items and other GUI controls in the application In fact the journal file written by the application to save its history is itself a Python script which can be run to regenerate the state of the application The journal file journal py is written to a user specific local directory on the com puter currently running the application On Microsoft Windows 2000 and XP the jour nal can be found in C Documents and Settings USERNAME Application Data OpenEye AFITT 1 3 On Microsoft Windows Vista the journal can be found in C Documents and Settings USERNAME AppData Local OpenEye AFITT 1 3 On all other platforms the journal can be found in USERNAME OpenEye AFITT 1 3 If the application were to unexpectedly exit the journal file corresponding to that run will be saved with a separate file containing a time stamp of when the application exited These journal files are particulary useful for debugging unexpected application behavior and are of extreme benefit to the application developers when tracking down user reported bugs The inclusion of journal files if possible is greatly encouraged when submitting bug reports 14 2 Startup File 118 14 3 Scripting Manual 119 Sometimes it is desireabl
150. o bond exists generate a double bond between the selected atoms 5 Triple bond change the selected bond to a triple bond or if no bond exists generate a triple bond between the selected atoms For each operation the number of hydrogens implicit or otherwise is correctly determined 86 Chapter 8 Protein Modeling 8 6 1 Adding Covalent Bonds Covalent bonds can be added by selecting an atom from the ligand and an atom from the protein If the atoms are in different molecules i e did not come from the same PDB file then a new molecule will be generated by combining the ligand and the protein with the appropriate bond The old protein and ligand will be preserved Bound ligands are added to a new chain id that does not exist in the protein If this cannot be done then the ligand is assigned a new residue number When adding a covalent bond the ligand atom names are regenerated so that the atom names do not clash with the protein and the atom names themselves are in the proper PDB style This has a side effect that the atom names specifically the atom ordering for the ligand might change For example atom O16 might become atom 05 8 7 Mouse Mode Toolbars The mouse mode toolbars change the behavior of the mouse in the 3D window The default behavior of the mouse controls selection and rotation of the main view Other mouse modes control altering residue and bond torsions translating the modeled atoms in 3D and modif
151. olecule Energy Molecular energy of the molecule as specified in the input file Actual Charge Sum of the partial charges on all atoms Formal Charge Sum of the formal charges on all atoms Halide Count Number of halogen atoms in the molecule Num Carbons Number of carbon atoms in the molecule Num Formal Charges Number of atoms with a specified formal charge Num Heavy Atoms Number of heavy atoms non hydrogen in the molecule Num Hetero Atoms Number of hetero atoms in the molecule Num Hydrogens Number of hydrogen atoms in the molecule Num Rigid Bonds Number of rigid bonds in the molecule Nom Rotatable Bonds Number of rotatable bonds in the molecule Num Chiral Atoms Number of chiral atoms in the molecule Num Chiral Bonds Number of chiral bonds in the molecule Table 11 2 Available molecular properties 11 6 Filtering New spreadsheets may be generated from existing one by means of filtering The mechanism to filter a spreadsheet is very similar to that of create a new column see Section 11 5 To create a filtered spreadsheet select the Filter Spreadsheet option from the top level Data menu This will launch a dialog which can be seen in Figure 11 5 This dialog will assist in the creation of a new filtered spreadsheet based on the currently viewed spreadsheet Please note that it is possible to create filtered spreadsheets on other filtered spreadsheets The result of a filtering operation can be seen in Figure 1
152. on of small molecule fragments over the protein While this can take some time the resulting potential field can be used as density where active site bounding regions can be manually selected as blobs as in section After creating a negative image the contour sliders can be used to increase or decrease the size of the located active sites Once a desirable volume has been set the manual blob picking process can be used to select blobs that will be used as bounding regions7 2 This process is time consuming for large proteins so be prepared to go out to lunch As the figure demonstrates SCiNI can get remarkably close to the protein s active site in this case the ligand is the crystallgraphic ligand and the detected region is the active site 7 8 Writing Refinement Dictionaries 7 8 Writing Refinement Dictionaries 77 Smery Meuse Sye Wircow Help z 10 tei HRA GERBERA 2 08 ALAC Result BETA LACTAMASE T Ay i or AW za os 7 Tec a 3 TecloNeaoee2W CCAA md Kl Figure 7 2 Shape Contour Negative Image AFITT has the ability to writeout refinement dictionaries suitable for REFMAC and CNS X PLOR To write out a refinement dicionary simply choose Write Refinement Dictionary from the file menu then choose the type of refinement program being used and finally choose the scope of the molecules being written from their respective submenus At this
153. operty of a single object in the application there can be only one Focused object at any given time Being Focused indicates that that particular object is the current object of interest and is therefore the primary focus of all relevant windows If the Focused object is not already Visible see Section 3 3 2 it will behave as if it were Visible for as long as it remains Focused In addition when a molecule is the Focused object its SMILES representation is displayed in the applica tion s status bar which can be found at the bottom of the application window If any of the molecule s atoms are selected that selection will be indicated in the SMILES display by the coloring of relevant atoms Selected atoms are colored using the current Selection Color the default is orange and are also in a bolded font while all of the unselected atoms will be displayed in their usual normal black font For large molecules gt 100 atoms in this case no SMILES representation is displayed as it would not fit 3 3 Object States 7 in the status bar 3 3 2 Visible State The Visible state is an indicator of whether or not the associated object is drawn in the 3D display see Chapter 5 Multiple objects can be Visible simultaneously If AFITT is operating in tiled mode each Visible object will be displayed in its own individual pane If the 2D display see Chapter 10 is also shown and AFITT is in tiled mode Visible molecules will be drawn in the
154. ortcut r Rotate Torsion Shortcut Ctrl b Rotate Torsion Backwards Shortcut Ctrl Shift B Rotate Shortcut f Rotate Y Shortcut s Rotate Carbonyl Rotate X Shortcut Rotate X Shortcut 2 Rotate X Shortcut 3 Rotate X Shortcut 4 A Rotate X Shortcut 5 Pa Rotate Backbone RRR RSENS Figure 3 11 Mouse Toolbar Most mouse modes have automatic monitors that show the torsion or rotations angle are being set Also if a density grid is available i e visible the rotated atoms displays a spherical halo the size of which corresponds to its placement in density Smaller halos indicate that the atom is not surrounded by much density while larger halo s indicate that an atom is surrounded by more density These monitors can be turned off from the Preferences menu 3 13 Protein Task 21 a Poor density fit b Good fensity fit Figure 3 12 Examples of visualization of poor and good density fits Noticed that in the poor fitting example the fitting halo in some of the ring atoms have disappeared e Residue Toolbar The residue toolbar is located at the bottom of the 3D Window This toolbar is designed to modify or append residues to the current protein If one or more residues are selected clicking on a residue in the toolbar will replace the currently selected residues If no residue is selected then a new residue is append to the end of the protein For exa
155. otations angle are being set Also if a density grid is available i e visible the rotated atoms show a spherical halo whose size corresponds to its placement in density Smaller halos indicate that the atom is not surrounded by much density while larger halo s indicate that an atom is surrounded by more density as seen in figure 8 6 a Poor density fit b Good fensity fit Figure 8 6 Examples of visualization of poor and good density fits Noticed that in the poor fitting example the fitting halo in some of the ring atoms have disappeared 8 8 2D Sequence View The 2D sequence view shows the protein sequence of the currently visible molecule If a ligand is Focused then it shows the 2D structure of the ligand Residues can be selected by clicking on the appropriate sequence 8 9 Ramachandran plot AFITT includes an interactive Ramachandran plot available from Window Ramachandran plot in the top menu The Focused molecule is depicted in the Ramachandran plot There are multiple Ramachan 88 Chapter 8 Protein Modeling dran plot modes available right click in the plot window to pop up a menu which allows changing the mode Richardson General Many 18 004 2 00 35 004 Psi N 80 00 0 080 7200 30 0 00 36 00 7200 10800 14400 18000 Phi Figure 8 7 Ramachandran Plot When a specific residue is selected only that residue and it s immediate neighbors are shown in the Ramachandran plot otherw
156. ould only be done if there is no expectation that the user loading the resulting state file will want to look at grid contour levels other than those already defined when the file was generated To save a state file select the Save State option in the File menu and specify the desired state file The state file version can be selected using the file format filter as seen in Figure 4 5 4 4 Importing Files External spreadsheet data can be imported into AFITT and associated with currently loaded objects Both comma separated files csv and tab delimited files txt are supported by default Once a file has 4 5 Exporting Files 3 been selected an import dialog will appear which allows the user to customize the column delimiters column headers as well as the criteria on which rows are matched to currently loaded objects For a more specific details on importing spreadsheet data please see Section 11 9 4 5 Exporting Files Multiple special file types can be exported from AFITT including spreadsheet data images and scripts Exact details about these files can be found in the relevant sections below 4 5 1 Data Spreadsheet data can be exported in two formats comma separated files csv and tab delimited files txt Data can be exported by selecting the Spreadsheet option in the Export submenu in the top level File menu Selecting this option will launch a file dialog which allows the user to specify the output file
157. ovides the primary user interface for performing modeling operations as well as changing visualization aspects such as slabbing control contour level and molecular style visualization The Style Control has several different widgets each of which controls a different aspect of visualization operations Most of these controls are described in Chapter 5 In addition to using the buttons in the the modeling window there are many keyboard shortcuts or hotkeys installed which make the most common modeling operations one keypress operations The hotkeys are active when the cursor is in either the 3D window or the modeling window When ap plicable hotkeys are given in the tooltip for the individual command button and in any menu item which references the given command The hotkey bindings may be changed by the user if desired See WindowRegisterHotkey in the scripting manual for details on customizing the hotkey bindings 82 Chapter 8 Protein Modeling The modeling widget is where the remainder of the modeling operations are placed Modeling operations include operations on bonds such as breaking and creating bonds residue operations such as creating or replacing residues and operations on rotamers such as locating the highest probabillity rotamers from the various rotamer libraries Even though AFITT includes a highly comprehensive Undo Redo facility sometimes it is useful to backup proteins and ligands during large scale changes such as auto
158. par and top files as appropriate There are three options to the dictionary creation e Include rotors By default constraints for free rotors torsions are generated as they facilitate manual modeling in such crystallography programs such as Coot Note that free rotor torsions are biased toward the current conformation of the molecule OpenEye recommends leaving this option on but if desired setting this parameter off will might be useful in some instances Command ModelingDictionaryRotors e Suppress Hydrogen By default any hydrogen atoms present are not included in dictionary pa rameters as most commonly the resolution of the data doesn t justify use of hydrogen atoms in the refinement e Aromatic model The aromatic model is important because aromatic ring systems are treated as planar and planarity constraints are generated for planar ring systems The default is the OpenEye OE aromatic model Other valid values are Daylight Tripos MMFF and MDL For more information on aromaticity see the OEChem docs 21 Note that the values set here are remembered as user preferences and will be remembered when restarting AFITT 6 3 Edit menu This section describes modeling related editing options located in AFITT s Edit menu 6 3 1 Copying Cutting and Pasting atoms e Copy This command copies all currently selected atoms even atoms from multiple molecules to an internal clipboard This clip
159. play charges in the calculation if any were specified However if no partial charges were specified AFITT will assign partial charges using either MMFF94 the default or AM1 BCC 19 20 The choice of charge model can be specified in the application preferences If a molecule has greater than 50 heavy atoms MMFF94 will always be used in place of AM1 BCC regardless of the set preferences Proteins have an alternative charging option based on residue information The charge model can be seen in the table below This model is used by default if no partial charges were specified but it can be disabled in the application preferences ASP 0D1 0D2 0 5 GLU 0OE1 OE2 0 5 LYS NZ 1 0 ARG NE 1 0 Other 0 0 Grid The grid scheme colors each vertex on the surface according to the grid potential at that vertex using a red to blue gradient from the minimum grid value to the maximum grid value The grid potential is determined by mapping the specified grid onto the surface Hydrogen Bond Potential The hydrogen bond potential scheme colors each vertex according to the hydrogen bond class of the nearest atom to that vertex in the molecule that the surface was created from see Figure 5 20 g This scheme will not work if the surface was not created from a molecule and also if that molecule is not currently present Hydrophobicity The hydrophobicity scheme colors each vertex according to a specified hydrophobic color scale based on resi
160. preadsheet see Chapter 11 and can be edited there as well as in the 3D display Annotations are saved as a SD tag when a molecule is written out in either SD or OEB formats 5 8 2 Bookmark The bookmark widget is a simple widget drawn at the bottom of the 3D display containing a clickable list of the currently available bookmarks Clicking on any individual bookmark will load that bookmark and update the display accordingly For more information on bookmarks see Section 5 5 5 8 3 Data Display This widget provides a simple table which contains the spreadsheet data of the Focused object The widget can be moved around the 3D display by clicking and dragging it to the desired location It can also be resized by clicking in the tab in the upper left hand corner Lastly if the amount of data associated with the object is too large to be shown all at once the widget does provide scrolling functionality 5 9 Molecular Visualization 49 5 8 4 Depiction Widget The depiction widget draws a 2D depiction of the focused molecule directly into the 3D display The depiction is drawn in the lower left hand corner of the display or individual pane if in tiled mode The depiction style is determined based on the application 2D depiction preferences The size and other drawing parameters e g anti aliasing of the depiction are also specified in the applicaton preferences 5 9 Molecular Visualization This section describes the very powerful ver
161. r 5 The display of peripheral windows is controlled by toggling the desired window options in the Windows menu Currently visible windows are indicated by the presence of a check mark next to their names Peripheral can also be hidden by clicking on the X in the upper right hand corner of the window As mentioned above the layout of the peripheral windows can be controlled by dragging the individual windows to their desired locations see Figure 3 3 3 6 Undo Redo 12 Chapter 3 Getting Started AFITT provides multi step undo and redo functionality of almost all operations Operations can be undone by selecting the Undo option in the Edit menu or by hitting Ctrl Z on the keyboard Either of these will undo the last operation performed This can be done repeatedly or the undo history can be viewed in the Undo History option the Edit menu which allows selecting how far back into the undo history to go Please note that while this menu only displays the 10 most recent operations the undo history can be much greater so feel free to revisit the Undo History option to go back further Operations that were just undone can be redone by selecting the Redo option in Edit menu or by hitting Ctrl Y on the keyboard Either of these will redo the last operation undone This can be done repeatedly or the redo history can be viewed in the Redo History option in the Edit menu which allows selecting how far into the redo history
162. r CPK plastic space filling models The CPKnew color scheme is a variant on the CPK color in that it uses a slightly brighter version of some of the colors The atom colors are listed below with the RGB values for CPK in the third column and the RGB values for CPKNew in the fourth column if different 52 Chapter 5 3D Display H White 255 255 255 He Pink 255 192 203 Li Fire Brick 178 34 34 178 33 33 B Cl Green 0 255 0 C Light Grey 200 200 200 211 211 211 N Sky Blue 143 143 255 135 206 235 O Red 255 0 0 F Si Au Golden Rod 218 165 32 Na Blue 0 0 255 Mg Forest Green 34 139 34 Al Ca Ti Cr Mn Ag Dark Grey 128 128 144 105 105 105 P Fe Ba Orange 255 165 0 255 170 0 S Yellow 255 200 50 255 253 0 Ni Cu Zn Br Brown 165 42 42 128 40 40 I Purple 160 32 240 Unknown Deep Pink 255 20 147 255 22 145 Element The element color scheme is the default color scheme used when coloring molecules Colors are assigned according to atomic number The actual colors used for a given element can be edited in the application preferences as seen in Figure 5 12 a Group I Group II Transition Metals Others Formal Charge White 255 255 255 Grey 180 180 180 Blue 0 0 255 Red 255 0 255 Green 0 255 0 Magenta 192 0 192 Yellow 255 255 0 Lime Green 170 255 0 Dark Red 170 0 0 Dark Orange 170 85 0 Cyan 0 255 255 Light Bl
163. r proteins an alternate residue based charging model is available and can be selected in the preferences This model assigns partial charges based on residue information as is shown below ASP 0D1 0D2 0 5 GLU 0OE1 OE2 0 5 LYS NZ 1 0 ARG NE 1 0 Other 0 0 The grid resolution is dynamically determined based on the number of atoms in the input molecule However it is possible to specify a fixed resolution in the grid preferences Be warned however that specifying very fine resolutions lt 2 0 Angstroms may consume a surprisingly large amount of memory and in some cases more than may be available on the computer which can lead to program failure This should be consider a very advanced feature to be used only when necessary and when all other data has been saved Finally it has been observed in research at OpenEye that adding salt to the electrostatic calculation has a good effect on the grids generated The salt concentration can be specified in the preferences The default concentration is 0 04 M The maximum concentration that can be specified is 0 1 M Electrostatic grids as well as ET grids are special in that they are always generated with two default contours a positive one and a negative one These contours cannot be deleted and new contours cannot be added The positive contour is colored blue by default and the negative contour is colored red The contour levels of the two contours are coupled together and so changing one will c
164. rch the DATA directory for any grid that matches grd any ligand that matches lig pdb gz and any protein that matches nolig pdb gz The matching rules are as follows e These wildcards are assumed to be relative to the expanded directory So if the directories that match the dir option resolve to a and b the ligands would be a lig pdb gz and b lig pdb gz e If multiple ligands grids or proteins are found in a given directory each will be fit in turn That means that if 2 grids match the wildcard 2 ligands match the wildcards and 2 proteins match the wildcards 8 independent fits will be done 19 2 2 Changing the output filename By default the automation system writes a file named result sdf to the directory being analyzed The reason that sdf is used and not pdb is so that wildcards searches will not pick up the result files but rather the original ligand pdb files To change the result name use the option outname this_is_my_new_name In this case the new output name will become this_is_my_new_name To change the extension supply one of pdb oeb sdf or oes Using an extension of oes saves a state file with all of the data used during the fitting processes and all of the results To change the output extension use the option outext oes This example will save state files as output If a file with th
165. rent With Parent if Mimics InOwnPane restore _appiy_J _ save _ cancer Figure 16 3 Preferences for 3D Display 132 Chapter 16 Preferences 16 1 3 Stereo Enumeartion By default AFITT only enumerates stereo for unlabeled stereo centers To force AFITT to enumerate all stereo centers whether they are labelled or not check the Enumerate Stereo option in the Ligand preference section as seen in figure 16 4 Additionally AFITT displays a text box to notify when stereo centers are missing To disable this notification uncheck the Notify Missing Stereo option Preferences Conformer Generation Shape MMFF Parameters Conformer Generation Options te O Enumerate Stereo Max Confomers Generated 30000 E neutralPh Max Conformers 30000 Notify Missing Stereo Window 25 000000 Conformer RMS 0 000000 Pool Size 30000 roo Ed GI Restore apply _ save __ Cancel Figure 16 4 Preferences for Ligand Fitting CHAPTER SEVENTEEN Example Data AFITT ships with an example data directory for the user to experiment with in the event that other input files are not immediately available Included in this directory are AFITT includes all of the protein ligand complexes in the GOLD data set that include structure factors These data sets are split into different directories each of which has the following files e protein mtz the protein only map generated through refmac5 e protein pdb
166. rface is at that vertex using a red to white gradient where red indicates a region of high concavity see Figure 5 20 c Curvature The curvature scheme colors each vertex on the surface according to the curvature of the surface at that vertex using a grey to green gradient where green indicates a region of high curvature see Figure 5 20 d Distance The distance scheme colors each vertex by its distance from either the selected set of atoms or the visible set of atoms if none are selected at the time of coloring Each band of color represents a distance of one Angstrom from the selected set The color banding is continuously repeated as the distance increases but the colors fade to white with each repetition to show the distance effect see Figure 5 20 e 5 11 Surface Visualization 6 g Color by Hydrogen Bond Potential h Color by Hydrophobicity Figure 5 20 Various different surface coloring schemes Electrostatics The electrostatics scheme colors each vertex on the surface according to the electrostatic potential at that vertex using a red to blue gradient from 7 0 to 10 0 see Figure 5 20 f This range can be changed if desired in the application preferences in the Surfaces section The electrostatic potential at the surface is calculated using OpenEye s Zap toolkit for more information please see the Zap toolkit documentation By default AFITT will use the molecule s input partial 62 Chapter 5 3D Dis
167. rites all files according to the standard however it does provide a mechanism to al low the user to override the handling of certain formats SMILES MDL SDF PDB Mol2 MF MOPAC and MacroModel The ability to change the flavor of a specific format can be done in the application preferences as seen in Figure 4 1 In addition to changing the flavor of a format there are a few other advanced options available when writing molecules including aromaticity model specification and conformer splitting The desired aro maticity model to be applied can be specified in the pulldown menu next to the Aromaticity Model label For more specific details on the available aromaticity models please see the OEChem documenta tion It is important to note that because these advanced options are available through the application prefer ences they will be remembed by the application and automatically applied in future Save operations unless they are subsequently restored 4 3 2 Grids Multiple grid formats are supported for writing and include e OpenEye grids e ASCII grids e GRASP Delphi grids 4 3 3 Surfaces Multiple surface formats are supported for writing and include e OpenEye surfaces e GRASP surfaces 4 3 4 State The entire state of a session can be stored in a single file called a State File oes A state file contains all of the molecules grids and surfaces that were loaded in AFITT at the time the state fil
168. rty Many miscellaneous 3D rendering improvements and accelerations Many miscellaneous user interface improvements New Features 10 Added dynamic help system Added new 2D depiction color schemes black on white white on black color on white and color on black Added new molecule coloring schemes amino chain group cpk cpknew shapely bfactor formal charge and partial charge Added new surface hydrophobicity scales charifson eisenberg kyte dolittle and white octanol Added support for reading attached grids and surfaces in OEB files Added support for reading Spicoli geometric annotations Added support for reading FRED receptor files Added new hydrogen style polar hydrogens only Added multiple new atom and bond label types including one for OEChem generic data Added new 3D display mouse maps for Coot and PyMol 21 3 Version 1 2 2 145 21 3 Version 1 2 2 July 2006 Minor Bug Fixes 1 Fixed MolecularEnergy calculation method in spreadsheet to correctly report energies on multi conformer molecules 2 Fixed SpreadsheetFilter command such that it operates on the specified spreadsheet instead of the current spreadsheet 3 Ensured that all current environment variables are properly passed to processes started from the process manager 4 Fixed the icon on the hydrogen display button to accurately reflect what will occur when the button is pressed 5 Fixed a Mac OS X b
169. s bar Similar to the Best Rotamer Search this uses the density in scope 84 Chapter 8 Protein Modeling If rotamers are changed or peptides are flipped the changed residues are colored gold to indicate that they have been changed and to make them easily visible in both the 3D Window or the 2D Preview e Fit To Screen Center AFITT s default is to use the density at the center of the screen as the target for optimization and fitting Normally this works well however there are some times where it is desirable to not use the screen center and use the current position of the selected atoms as the density target This toggle is seen in the panel shown in Figure 8 4 8 5 Appending Prepending or Mutating Residues or Rotamers Residues can be mutated appended or prepended to the current model in a variety of different confor mations As seen in Figure 8 4 the arrows select the previous and next residues while the buttons simply perform the selcted operation based on the residue type and geometry selected in the pull down menus Figure 8 4 shows the break down of the residue operations Operate at screen center ddr pipas P Next Residue Previous Residue E g E S Aa Add to C terminus M Alpha Helix E Add to N terminus E Parallel Sheet Mutate Selected Bm Anti Parallel Sheet Hi Create New H 3 10 Helix MA Pi Helix E Collagen Helix F Type II Helix Perform Residue Operation ajm slo lt r
170. s made to the options at the top Once the preview of file to be imported is satisfactory click on the Next button to specify how the rows are to be matched to currently loaded molecules and what to do with rows that do not match see Figure 11 9 11 9 Importing Exporting 105 Figure 11 9 Spreadsheet Import Dialog Spread Sheet Import Match Rows By Import unmatched rows List Order cox2_good sdf gz By SMILES y O Name Name Y O smiLES Name Y O Values in Name O Do Not Match Apply Function To Column Ignore 1C50_uM 0 9600 0 9500 0 9400 0 9200 Cancel Prev Finish There are a number of mechanisms available to match data rows to the appropriate molecules in AFITT e List Order matches the order of rows in the spreadsheet with the order of molecules in the selected list e Name matches the molecule s name with the name in the selected column of the imported data e SMILES matches the molecule s canonical SMILES string with the SMILES string in the se lected column of the imported data e Values In matches the value in the selected column between the imported data and the value already in the spreadsheet e Do Not Match assume that you are importing new molecules Unmatched rows can also be imported into the spreadsheet by creating a new molecule in AFITT to associated with that row The following options are available for unmatched rows e By SMILES use the SMILES
171. s starts and is saved back to disk when the application exits Deleting this file is equivalent to clicking on the Restore button in the dialog box There is also a file in this same directory which called AFITT ini which contains machine specific settings like the list of recents files prefered layouts and hardware stereo options Deleting this file will restore these settings to the defaults as well This directory can be opened from within AFITT by selecting the Open User Directory menu item in the Help menu 130 Chapter 16 Preferences 16 1 Useful Preferences A brief list of useful preferences and their locations is given These preferences are particularly useful for customizing AFITT for ligand fitting 16 1 1 Display Hydrogens Preference By default only polar hydrogens are shown While changing the hydrogen display in the style control is possible this only affects the hydrogens in scope By changing the default hydrogen prefence from Polar to All all explicitly defined hydrogens will be shown when loading new files This preferences is available in the Molecules section as seen in figure 16 2 Preferences Styles 2D Depiction Small Molecules Stick Smal Cylinder Aromatic Circles Y Aromatic Dashes Large Molecules Wireframe None Wireframe Dative Bonds O Bow Ties Methyl C super Atoms Cutoff of atoms 255 El meth C Hycrogens a Color on Black Color on White Show Non Bonded Atoms Show Aromatic
172. satile and interactive molecular visualization capabilities of the 3D display The interactive control of molecular visualization is typically performed using the Style Control see Section 5 1 2 or through scripting commands for more details on scripting see Chapter 14 5 9 1 Display Styles All of the standard display styles are available Wireframe Stick Ball and Stick CPK and Stars and can be seen in Figure 5 11 By default small molecules are drawn in Stick mode while large molecules are drawn in Wireframe mode These defaults can be changed in the application preferences see Chapter 16 59 2 Color By default the atoms in a molecule are colored according to their element types and the bonds in a molecule are colored according to the element types of the two atoms defining that bond There are two color palettes that specify the actual colors used for a given element one for use with dark colored backgrounds and the other for use with light colored backgrounds Both of these palettes can be viewed and modified in the application preferences see Chapter 16 The dark background atom color palette can be seen in Figure 5 12 a Coloring Schemes There are a number of molecule specific coloring schemes in addition to the standard element based coloring The following schemes are discussed below amino bfactor carbon chain cpk cpknew element formal charge group partial charge reference residue and shapely Amino
173. sigma value for the density Selecting the Auto checkbox forces the searching algorithm to examine all density sigmas in an attempt to find a blob of the appropriate size Finally once a blob has been selected clicking on the Fit button will begin the fitting process When complete the fitted small molecules will appear on the screen and in the spreadsheet 7 2 3D window 73 7 2 3D window Most of the time spent in AFITT will be spent in the 3D viewing window which is the largest area of the application This window shows the currently visible objects such as density grids molecules proteins and blobs Left clicking on most items will select them For instance left clicking on a blob will select the blob and make it focused The selected blob will be used to extract the density region that will be used to fit the small molecule 7 3 Spreadsheet The spreadsheet located at the bottom left as seen in Figure 7 1 shows more details about the objects that are loaded in AFITT It is seperated into five tabs Molecules Proteins Maps Blobs and Results The Molecules tab holds the small molecules that are loaded into AFITT and the The Proteins tab show the proteins loaded into AFITT The Maps tab contains the currently loaded densities and grids Blobs contain the automatically located blobs and surfaces Finally the Results tab hold the small molecules that have been fit to the density For more information about using the spreadsheet se
174. string in the selected column of the imported data to create a new molecule e Apply Function Apply a Python function to the data in the selected column of the imported data to create a new molecule This can be particularly useful in cases where one of the columns contains a database ID which can be used to retrieve molecules e Ignore Do not import unmatched data 11 9 2 Exporting Spreadsheets may be exported to an external file by selecting the Spreadsheet option in the Export submenu of the top level File menu Selecting this option will launch a dialog which allows for specifica 106 Chapter 11 Spreadsheet tion of which columns to export see Figure 11 10 Supported formats include either comma separated csv or tab delimited txt Figure 11 10 Spreadsheet Export Dialog Spreadsheet Export Spreadsheets All Columns VIDA Name All except Lists VIDA ID Molecules Name Proteins Family Atoms 1 50_uM 1C50 E CHAPTER TWELVE List Management The organization of molecules into lists and the ability to easily browse through these lists is an extremely important and central concept to AFITT As such a List Window is provided to aid in the navigation and organization of lists Furthermore there exist many mechanisms to create modify and delete lists which will be discussed in more detail later in this chapter 12 1 Introduction The List Window is one
175. t Window displays which objects are currently loaded in AFITT along with their visibility and marked states e 3D Window As in the Ligand Task the 3D window takes up a majority of the view and controls most of the action Changes to protein structure and protein fit to density are indicated here Atoms bonds and or residues may be selected by left clicking or left double clicking 3 13 Protein Task 19 e Style Bar and Modeling Widget MMFF Shape Fit Selection Shape and Torsion Fit Mark State Regularize Selection Revert to last mark BeA Anaon x l Clean Up Protein Rigid Shape Fit Selection Pep Flip Find Best Rotamer Figure 3 8 Protein Controls Current Residue Next Residue Previous Residue EN oa P aLa323 gt Operate at screen center El Add to C terminus M Alpha Helix E Add to N terminus E Parallel Sheet Mutate Selected Be Anti Parallel Sheet Hi Create New H 3 10 Helix MA Pi Helix E Collagen Helix F Type II Helix Perform Residue Operation ajm slo lt j lt lt ri gt K z z nio vIn mio Figure 3 9 Residue Controls The modeling widget is contained at the top of the Style Bar The Style Bar contains many different components that can be used to alter view settings control density and more For a complete description of the Style Bar please see chapter 5 The modeling widget is where the remainder of the modeling functionality is lo
176. t was running 13 Reorganized Edit menu for more logical placement of options as well as to provide some com monly performed operations in a single place a Moved Split Molecules functionality to File menu b Added new Select submenu which contains the following options All Visible Query Invert and None c Added new Mark submenu which contains the following options All Query None d Added new Hide submenu which contains the following options All Selected Marked Not Selected Not Marked and None e Removed the following options as they were redundant given the above additions Hide All Clear Marked 14 Alphabetized list of available windows in the Window menu 15 Batch shell window no longer appears when running on Windows 16 Redesigned process manager window for easier use 17 Significantly improved the quality of POV ray exports and reduced the output file size 18 Improved the rendering of electrostatic grids New Features 21 4 Version 1 2 1 147 1 2 Added Copy Image function under the Edit menu which takes a screenshot of the current main window and places it on the clipboard Added Map Index atom label corresponding to the value returned by the OEChem function OEAtomBase GetMapldx 21 4 Version 1 2 1 March 2006 Minor Bug Fixes 10 11 12 13 Fixed a problem where extra atom labels could appear on molecules when saved to state Fi
177. tandard mtz files output by programs such as refmac3 and other crystallography appli cations The first time an mtz file is opened AFITT will prompt for the columns to use when loading A description of these columns can be seen in Chapter 19 2 5 This prompt with the default columns for refmac5 can be seen in figure 4 2 Once the columns are chosen the type of maps to generate need to be decided AFITT only displays map choices that can be created from the columns This dialog can be seen in figure 4 3 AFITT remembers the last type of file opened and will attempt to choose the same columns for the next mtz file If this succeeds the file type prompt will not be shown and the maps will automatically be created 4 1 4 Surfaces Multiple surface formats are supported for reading and include 26 Chapter 4 File I O Set MTZ Column na 9 IDELFWT PHDELWT SIGFP Figure of Merit FOM v FreeR_flag y RFree FreeR_fag la Figure 4 2 Column choosing for mtz files Calculate maps 9 x Regular map A v Difference map Fdeiwt la Jl coma Figure 4 3 Map type chooser for mtz files e OpenEye surfaces e GRASP surfaces 4 1 5 Python Scripts AFITT supports reading of Python scripts compatible with Python version 2 3 AFITT supports two ad ditional file extensions for Python scripts pyv and vpy to enable users to associate those file extensions with AFITT without adversely affecting the
178. the currently modeled residue If density is currently in scope this chooses the library rotamer that best matches the density If no density is being modeled this chooses the highest probability rotamer Pepflip Shortcut v Flips the peptide backbone Shape Fit Shortcut d This performs a rigid body shape fit Most of the time this produces un acceptable results since the rotamers are not allows to change to match the surrounding density In some cases this causes the residue to find a slightly distant portion of density that the current geometry prefers which in turns causes ridiculously long bond angles Cleanup Protein Cleans up the entire protein on a residue by residue basis The following dialog appears when clicked Cleanup Protein Targets Protein 1428 pdb ligand PROGESTERONE RECEPTOR Map Fdelwt 1428_prot mtz Options Cleanup Rotamers Cleanup Pep flips C MMFF Shape Fit Residues Figure 8 3 Clean protein dialog The current target proteins and map is shown if these are not the desired targets simply cancel the dialog and make the desired protein and map and only the desired protein and map visible This dialog can be used to adjust which aspects of the protein are being cleaned The available options are Cleanup Rotamers Cleanup pep flips MMFF Shape Fit Residues After the the cleaning process is started it can be canceled at any time by clicking on the Stop button next to the progres
179. the desired file The file formats listed in the file dialog filter will reflect which file formats are supported based on the objects selected Only the OpenEye binary format supports writing multiple object types molecules grids and or surfaces to the same file 28 Chapter 4 File I O Select ae ag aig Select S cox2_good sdf gz 1 16 cox2 1 17 cox2 1 18 cox2 1 19 cox2 1 20 cox2 1 21 cox2 1 22 cox2 1 26 cox2 1 27 cox2 1 28 cox2 1 29 cox2 x Figure 4 4 Selecting what to save This process can be expedited by simplying right clicking on the desired objects and selecting the Save option This allows the user to bypass the selection dialog and go directly to the file dialog 4 3 1 Molecules Multiple molecular file formats are supported for writing and include e OpenEye binary format v2 oeb e MDL RDF and SDF e Tripos MOL2 MOL2H e Daylight SMILES canonical SMILES and isomeric SMILES e ChemDraw CDX e MacroModel Molecular Formula MF MOPAC FASTA e PDB e XYZ 4 3 Saving Files 29 As is often the case with file formats the meaning and use of certain fields within a format may change over time which can potentially lead to problems interpreting those files VIDA using OpenEye s OEChem toolkit w
180. ting consists of 1 Loading the density ligand and protein 2 Finding the regions of density a k a blobs that are indicative of the ligand density 3 Fitting the ligand to these regions In most cases these three steps are all that are necessary for ligand fitting Advanced usage consists of 1 Adding constraint boxes for ligand fitting 2 Automatically identifying the potential binding sites and fitting to these regions 3 Manually selecting pieces of density to fit against The introductory screen for the Ligand task is seen in Figure 7 1 AFITT can automatically locate the appropriate density for the ligand In most cases all that is needed is loading the appropriate elements and then clicking on Find Blobs and then Fit The Model window organizes the grids and molecules used in ligand fitting and directs the workflow to the final fit The main interface elements that implement the ligand fitting task are the Ligand Modeling Widget the 3D Window and the Spreadsheet 7 1 Ligand Modeling Window This window located on the left side of the application as seen in Figure 7 1 shows the state and basic operations available for fitting small molecules to density To fit a small molecule to density it is nec 71 72 Chapter 7 Ligand Modeling File Edit Task Data Style View Window Help Focused visble marked al M HHS Model Ligand Map Fut 128_prot mtz Ligand cala Protein ligand PROGESTER
181. tion mechanisms as described in Section 5 1 1 The second button with the info icon changes the behavior of the mouse such that an informative label is displayed on the screen regarding whatever is currently underneath the mouse cursor The remaining three buttons put the mouse into one of three measurement modes Distance Angle and Torsion respectively More details about measurement and the monitors generated by the measurements can be found in Section 5 9 4 This toolbar can be reference in scripting commands using the name Mouse 5 7 3 Application Toolbar In addition to providing 1ts own toolbars the 3D display is affected by a few of the buttons in the main application toolbar The application toolbar contains the Tiled display toggle button matrix icon the main window screenshot button camera icon the centering button four arrows pointing in and the fitting button four arrows pointing out The Tiled display button toggles the display between single pane overlay mode and multiple pane tiled mode For more details see Section 5 6 The screenshot button captures an image of the current main window at a user specified resolution The centering button centers the display on the currently selected 5 8 Display Widgets 47 set or whatever molecule s are in the current scope The fit button centers the scene and adjusts the scale factor such that all Visible objects can be seen on the screen at the same time 5
182. tton mouse For users not comfortable or familiar with the mouse interactions described below AFITT can be made to emulate the mouse behavior of many other applications including Coot Insight Maestro MOE O Quanta PyMol RasMol and Sybyl The desired mouse map can be set in the application preferences in the 3D Display section Please note that not all the functionality described below is available in the emulated mouse modes nor is all of the functionality of the emulated applications available in AFITT Selection 34 5 1 User Interaction 35 Objects in the display can be selected by clicking on them using the Left mouse button The selection is cleared between button clicks or by clicking in the background unless the Shift or Ctrl key is also held down when clicking If a single vertex on a surface has been selected holding down the Shift key and selecting an second vertex will select the shortest path of vertices between the two selected end points Double clicking using the Left mouse button expands the current selection to the next logical grouping For instance double clicking on a selected atom in a protein will expand the selection to include all of the atoms in the same residue as the original selected atom Double clicking on the selected set again will expand the selection to include either the entire chain if there are multiple chains otherwise it will include the entire molecule Selection can also be performed usin
183. uated on row 1 of Molecules c0s takes exactly 1 argument 0 given 0 673529444142 a Creating a new column b Using a math function in the c Referencing another column expression Figure 11 4 Creating a new column from an expression New columns can be added to the spreadsheet containing values that are based on or calculated from molecular properties or existing spreadsheet data To create a new columns select the Create Column option in the top level Data menu This will launch a dialog to guide the process and can be seen in Figure 11 4 The text entry field at the top contains the name for the new column A new empty and editable column can be easily be created by supplying a new column name and then clicking the Create button Beneath the name is a collection of three drop down boxes containing functions which can be used in building the expression to be evaluated when populating the column The first drop down box contains a collection of available mathematical functions which can be used in the expression see Section 11 5 1 for more details The second drop down box contains a collection of available molecular properties that can be used in the expression see Section 11 5 2 for more details The last drop down box contains a list of the other already existing columns which can be used as part of the expression Any of the above described functions can be added to the expression simply by selecting them in the
184. ue 160 160 255 Purple 170 0 255 Pink 255 0 128 The formal charge color scheme colors atoms according to their formal charge using a fixed red to blue gradient between 4 and 4 Group 5 9 Molecular Visualization 53 The group color scheme is a macromolecule specific scheme which colors each atom according to its position in a macromolecular chain The colors are assigned along a smooth rainbow spectrum from blue to green to yellow to orange to red Partial Charge The partial charge color scheme colors atoms according to their partial charge using a red to blue gra dient between 1 0 and 1 0 Reference The reference color scheme colors all of the carbon atoms in the current scope using the current Reference color which can be set in the application preferences The default is green Residue The residue color scheme is a protein specific scheme which colors atoms according to their individual residues The actual colors used can be edited in the application preferences as seen in Figure 5 12 b The default colors correspond to those used in the shapely color scheme Shapely The shapely color scheme is a protein specific scheme which colors atoms according to their individual residues This scheme is based upon Bob Fletterick s Shapely Models 17 The residue colors are listed below 54 Chapter 5 3D Display ALA Medium Green 140 255 140 GLY White 255 255 255 LEU Olive Green 69 94
185. ug where double clicking on a molecule file started VIDA but did not actually load the file that was clicked on Major Bug Fixes 1 Upgraded to OEChem 1 4 1 which fixed a number of bugs associated with handling bad input files 2 Fixed a cross platform compatibility issue in state files Improvements 1 Automation AFITT now has easy to use automation for fitting ligands to density Please see Chapter 19 for full details 2 Box Files AFITT can now generate boxes to constrain the locations that ligands can be fit to density A box can be generated from any molecule or selection of atoms The bounding box of the molecule plus a specified padding is used to constrain the density in which to find blobs Alternatively the density inside the box can be used for fitting This is located in the Data menu 3 Mask Density To Blob The Ligand Model Window has a new button This button extracts the densities within each blob These new densities can be saved to disk or used for other operations 4 Write Refinement Dictionary If you are writing a refinement dictionary from a non 3D molecule say a smiles string a low energy conformer will now be generated for the molecule 5 Project List During ligand fitting all seconday objects like extracted grids and blobs are now put in the same list for easy access int the list window A new list is created when Find Blobs Blobs is clicked 6 Blob List Pressing the up and down arrows
186. usage for each task is as follows 3 12 Ligand Task The initial task displayed when opening AFITT for the first time is the Ligand task The basic workflow for ligand fitting consists of 1 Loading the density ligand and protein 2 Finding the regions of density a k a blobs that are indicative of the ligand density 3 Fitting the ligand to these regions In most cases the three steps described above are all that are necessary for ligand fitting AFITT s initial screen is the Ligand task seen in Figure 3 5 This workflow is divided into four sections described below 14 Chapter 3 Getting Started File Edit Task Data Style View Window Help Focused visble marked al M HHS Model Ligand ax Map Fwt 1428_prot mtz Orm Ligand cala Protein ligand PROGESTERONE RECEPTOR w Auto Find Y Show Modeled 1 000 risa o VIDA Name volume pr e a EA y a a AXE oO Molecular Volume 174 269 Model Ligand List Window Spreadsheet X 2DPreview 1428 pdb olal VIDA Name VIDA ID 1 0 1428 pdb ligand 8 Molecules Proteins Atoms Maps Blobs Results Spreadsheet Scripting Window CC 0 C H 1CC C H 2 C 1 CC C HJA C HJ2CCC4 CC O NCCLC B4C C Figure 3 5 Ligand mode layout 3 12 1 Ligand Modeling Controls The Model Ligand Window located on the left side of the application shown in detail in figure 3 6 shows the state and
187. uture versions of AFITT will enable the import of the full PyOEChem module which will allow modifications provided that a separate license for the OEChem Python wrap pers has been obtained Using cecheml ite a molecule is accessed by calling the ScriptableObjectGet function which takes an ID as a parameter and returns the associated molecule For more details on the associated API please see the PyOEChem documentation however please remember that only the const functions those that do not make changes to their objects are available in oechemlite As most scripting routines in AFITT take unique keys as parameters a Get Key function has been added to every object which returns the associated object s unique key identifier In addition the following methods have also been added to all oechemlite objects for convenience 1 IsActiveQ 2 IsMarked 3 IsLocked 4 IsSelected 5 IsVisible 6 SetLocked value True 7 SetMarked value True 8 SetSelected value True 9 SetVisible value True Two example scripts which use oechemlite appear below def SelectCarbonAtoms idOrKey Select all carbon atoms from the given id or key if an id is used only carbons from the first conformer are selected PushIgnoreHint 14 5 OEChemLite 12 try mol ScriptableObjectGet idOrKey mol SetVisible for atom in mol GetAtoms if atom IsCarbon atom SetSelected finally the try finall
188. visualized in AFITT The area beneath the dividing line is a collection of buttons whose functionality is specific to just molecules In the top area the first button with the molecule icon contains a drop down menu of the available molecule display styles ball and stick CPK stars stick wireframe and hidden Selecting one of these AO Chapter 5 3D Display k 55 X EI AGN H Hp Figure 5 6 Style pane of the Style Control options will change the display style of all the atoms and bonds in the current scope For more details on these individual styles see Section 5 9 1 The second button with the surface icon contains a drop down menu of the available surface display styles solid mesh and points Selecting one of these options will change the display style of all the surfaces in the current scope For more details on these individual styles see section 5 11 1 The third button with the grid icon contains a drop down menu of the available grid display styles solid line and cloud Selecting one of these options will change the display style of all the grid contours in the current scope For more details on these individual styles see Section 5 10 The fourth button contains a drop down menu of the supported grid types in AFITT Electrostatic ET FRED Generic Difference Map and Regular Map Selecting one of these options will change the grid type of all the grids in the current scope A grid s type determines how
189. w 81 File Edit Task Data Style View Window Help Focused visble merked al I BH List Window ax Style Name 0270 A Modeling 1428_input pdb E EJ a e L as ogpgogogggaama WME I 10 10 39 999 y canta Regular Map 2Fwt 1428_prot mtz Difference Map None 5 1428 pdb ligand 6 2 1428_afitt_lig pdb 7 1428 pdb ligand PR 8 Color aer Sao Transparency 0 X Ramachandran Richardson General QLI PPLINLIMSIE PDVIYAGHDNTKEDT SSSLLTSLNOLGERQLLSVVEWSKSLPGERNLHIDDOITLI Gy SWwMSLMVE GOGWRSYKHVSGOMLYFAPDLILNEORMKE SSE 4 5 O POPE VETS P EEVEE EEE LOMEVLLLINTL PLEGLRSOTOFEEMRSSYIRELIKAIGLROKGVVSSSORFYOLTKLLONLHDLVKOLHLYCINTFIQSRALSVEF PEM LIPPLINLIMSIEPDVIYACHONTKEDTSSSLLTSINOLGEROLLSVVKNSKSLPEGFRNLHIDDOITLIQY SWMSLMVE GLGWRSYKHVSGOMLYF AEDLILNEORMKESSE 85 SLOT POEEVELOVSCEEFLCMKVLLLINTI BLE GLRSOTOF EEMRS ST RELIKALGLROWVVSSSORE YOLTRLLENLHDLVRQLHLY CINTELOSRALSVEE FEM b10 SEVIAAOLPRT 2D Preview Scripting Window CC 0 C H 1CC C H 2 C 1 CC C H 3 C H 2CCc4 CC Oj CC C 340 C Figure 8 1 Protein Workflow Layout 8 2 3D Window As in the Ligand Task the 3D window takes up a majority of the view and controls most of the action Changes to protein structure and protein fit to density are indicated here and atoms bonds or residues may be selected by left clicking or left double clicking 8 3 Style Window The Style window pr
190. while inside the blob list will go to the previous or next blob respectively 146 Chapter 21 Release Notes 7 Retrieve Density now available on windows The option to retrieve density from the EDS server under the Data Menu is now available 8 Copy Paste Now work on focused visible molecules when not in modeling mode When inside modeling mode they work on modeled items as they did before 9 Mouse Modes When mouse modes are being used the current mouse mode is shown at the top of the 3D window 10 Updated menu system to be compliant with standard UI design practices such as usage when menu option launches a dialog 11 Updated menu system on Mac OS X to follow common Mac paradigms e g Open Recents and Preferences options 12 Reorganized File menu for more logical placement of options a Collapsed New gt List gt submenu structure to a single New List gt submenu which appears after the Open Save block b Added New Molecules gt submenu which contains a From Split submenu which pro vides the Split Molecules functionality from the old Edit menu In VIDA 2 2 a From Merge will join this option c Renamed Clear All option to Clear and moved up in the menu above Save block d Removed Abort Script option as it was redundant Stop button next to progress bar per forms this function and also could generally not be accessed while a scrip
191. xed a problem where CTRL mouse over atom displays were not seen when using non VIDA mouse maps Fixed a bug in the Text Scale slider where its position did not always reflect the current scale being used Fixed a bug where OE_ DIR and OE_ LICENSE environment variables were not being properly exported to the Process Manager on Mac OS X such that spawned applications could not find a valid oe _license txt file Fixed a bug where exporting a CSV file on Microsoft Windows attached an extra return character causing a blank line to appear between each row when read into Microsoft Excel Fixed a few cases where the display of depictions in the spreadsheet was not obeying the set preferences Fixed a bug where extra calls to undo cleared the redo stack Turned off the writing of empty data fields when writing molecules to SD or OEB formats This can be turned back on if truly desired from the Preferences Fixed a number of preferences that were not being properly saved or obeyed by the application Fixed the undo stack such the initial setup of the 3D display cannot be undone by accident Fixed the display of angle monitor measurements from radians to degrees Fixed the display of the text scale widget such that it is not hidden on low resolution monitors Made sure that when saving images and state files a proper file extension is added to the filename even if one was not specified Major Bug Fixes 148 Chapter 21
192. y will always call the popignorehint even if there is an error otherwise the application might not work correctly PopIgnoreHint def CreateSphereMonitorAtomsByDistance idOrKey x y z distance idOrKey X Y Zz distanc gt generate sphere monitors for all atoms belonging to the specified id or key with distance from coordinate x y z When using an ID only atoms from the first conformer will be examined distanceSquared distance distance PushIgnoreHint True try mol Scriptable0bjectGet idOrKey mol SetVisible color OEColor 255 255 0 100 for atom in mol GetAtoms x1 y1 z1 mol GetCoords atom dx x xl dy y yl dz z z1 if dx dx dy dy dz dz lt distanceSquared CreateSphereMonitor atom GetKey Within Distance color finally the try finally will always call the popignorehint even if there is an error otherwise the application might not work correctly PopIgnoreHint Please note that it is not necessary to import oechemlite to access this functionality that is done automatically at startup CHAPTER FIFTEEN Query language 15 1 Introduction AFITT has several built in scripting commands such as Visible Select Lock Mark and Subset which take a string argument see also Section 15 6 where that string is written in a query language This query language is somewhat similar to the command language of the molecular graphics programs GR
193. ying and placing the c alpha backbones Hold the mouse over each button to get a tooltip describing the various modes 1 World Mouse Shortcut w Sets the mouse to the default mode 2 Translation Shortcut t translate the selected atoms in space 3 Rotate Selected Center of Mass Shortcut Shift R rotate the selected atoms around their center of mass 4 Rotate Selected C Alpha Shortcut r rotate the selected atoms around the residues C alpha carbon 5 Torsion Shortcut CTRL B Rotate around a torsion keeping one side of the torsion fixed 6 Reverse Torsion Shortcut CTRL SHIFT B Rotate around a torsion keeping the other side of the torsion fixed 7 Phi Shortcut f Rotate the Phi torsion of a residue 8 Psi Shortcut s Rotate the Psi torsion of a residue 9 Carbonyl Rotate the residues carbonyl group 10 Chil Shortcut 1 Rotate the residues Chil torsion 11 Chi2 Shortcut 2 Rotate the residues Chi2 torsion 12 Chi3 Shortcut 3 Rotate the residues Chi3 torsion 8 8 2D Sequence View 87 13 Chi4 Shortcut 4 Rotate the residues Chi4 torsion 14 Chi5 Shortcut 5 Rotate the residues Chi5 torsion 15 Backbone Rotate the residue s backbone This mode rotates the residue s c alpha carbon around the previous residue s c alpha carbon and is useful for placing the backbone c alpha into appropri ate density Most mouse modes have automatic monitors that show the torsion or r

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