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Protein X-ray Crystallography Methods

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1. Group b factor minimization This process refines the b factors for side chains and main chain atoms as two separate groups The minimization is carried out by the CNS module bgroup inp Required inputs are the pdb mt f and cv files the unit cell parameters and space group any extra top or par files required the resolution range of the reflection data to be used typically all of the data and the output file typically bgroup pdb Individual b factor minimization This process refines the b factors for each individual atom separately The minimization is carried out by the CNS module bindividual inp Required inputs are the pdb mt and cv files the unit cell parameters and space group any extra top or par files required the resolution range of the reflection data to be used typically all of the data and the output file typically bindividual pdb A minimization cycle consists of running minimize inp bgroup inp and bindividual inp in that order After each cycle new 2F F and F F maps should be generated from the final output file bindividual pdb It is a good practice to copy or rename this file to indicate the identity of the crystal and the number of refinement cycles For example after the 2 refinement cycle of the crystal HICA 08 the pdb file might be named hica08 2 pdb A simple Linux script for automating a refinement cycle is shown in File 10 The script files for the CNS modules are ar
2. To merge datasets the second and subsequent datasets must be renumbered so that batches of reflections collections of reflections from a frame of data will have unique non conflicting batch numbers The resulting sorted datasets are then combined and sorted by reflection and then finally re scaled to render them consistent with each other The following procedure is typical for merging and scaling two data sets e Open a CCP4i session as previously described previously e Select the Data Reduction module in CCP4i upper left menu bar and click on Sort Modify Combine MTZ files A task window will open Figure 10 SA Sort Reindex MTZ Files Job title zm ber _j Change space group and or reindex reflections W Reset the batch number s MTZ in d44n01 44n01 highres_scalal mtz Browse u ew MTZout d44n01 44n01 highres_sort1 mtz Browse View a Edit Batch Numbers w eee Sorting Details Sortin ascending order of H K L Edit list Add a batch edit Save or Restore Close Figure 10 Sort Modify Combine MTZ files task window Mandatory fields are highlighted in color e Enter a job name e g renumber and select the appropriate MTZ input filename An output file name will be generated or you can change it to something else e Select Reset the Batch number s and enter a number for the first batch This number should be larger than the highest batch frame number in the b
3. First the files should be concatenated using a text editor any remark files can be removed Next the file should be reformatted so that each protein chain has a different SEGID This is most conveniently accomplished in MOLEMAN2 To invoke the program type moleman2 at the prompt To read in the PDB file type RE filename pdb where filename pdb is the concatenated PDB file To rename the segid s type CH AS and name the segid s A B C D etc At this point it is also useful to set the b factors to the value estimated by truncate To change all the B factors type BF LI and enter the b factor value from truncate as both the high and low limit To save the changes to the file type WR newfile pdb where newfile pdb is the new filename of the modified PDB file MOLEMAN2 is a very powerful program for modifying PDB files and is well worth learning more about Finding molecular replacement solutions using Phaser Phaser is another powerful molecular replacement program that is now integrated into the latest release of CCP4 Data that cannot be solved by EPMR can often be solved by Phaser and vice versa Phaser is most conveniently run via CCP4i and one feature of the CCP4i task window can be used to estimate the number of protein molecules present in the asymmetric unit prior to running either Phaser or EPMR Estimating the number of protein molecules in the asymmetric unit A utility within Phaser can utilize Matthews Probability calculations
4. This command is used to designate a shortcut name for a complicated command For example the command alias project10 cd projects project10 refine ncs would allow you execute the long complicated directory change in single quotes by simply typing project10 at the prompt Any valid Linux command can be placed within the single quotes Additional Resources e Beginner s Linux Guide http www linux ie newusers beginners linux guide e A Beginner s Guide to Linux http www geocities com aboutlinux Roger Rowlett 23 X ray Diffraction Data Analysis There are many software suites that can be used to analyze protein X ray diffraction data Described here is data analysis procedures based on the programs DENZO and SCALEPACK or MOSFLM and SCALA Indexing the first frame using DENZO When screening crystals for diffraction or before analyzing an entire data set it is necessary to index the reflections observed in the first frame in order to obtain the exact orientation of the crystal with respect to the X ray beam and to make a preliminary determination of the space group and unit cell size The programs XDISP and DENZO are used together to perform reflection indexing The following procedure is typical for indexing a single frame of data e Copy your image file s typically osc into a local working directory e Display a frame by issuing the command xdisp raxis4 100 filename osc amp where filename osc is the name of y
5. and must be read in before O starts Files 12 16 next_ca centers screen on next alpha carbon and redraws electron density maps as defined in on_startup centre_next atom _ name ca fm_draw 2fofc fm_draw fofc fm_draw fofc next_water centers screen on next solvent molecule and redraws electron density maps as define in on_startup centre_next atom_name o fm_draw 2fofc fm_draw fofct fm_draw fofc redraw_map redraws 2fofc and fofc maps as defined in on_startup fm_draw 2fofc fm_draw fofct fm_draw fofc redraw_solv redraws solvent molecules and protein useful after using add_water command rename solv and hica as required mol solv zo end mol hica gen_symmetry generates nearby lt 10 A symmetry atoms rename molecule hica and alter radius as required sym sph hica sym 10 0 60 X Ray Crystallography Methods Files 17 18 resid odb ID_TEMPLATE BK Restyp SRESNAM SATMNAM residue_2ry_struc odat odb edit deirectory to point to O data files odat t 1 50 C o data The on_startup file if present in the working directory controls what commands and other functions O should perform each time the program is started Normally on_startup should not read in any pdb files but it is useful for it to read in and format electron density maps menus and any O databases desired An example is shown in File 18 which reads in a custom menu the file re
6. Choose an output file name typically filename sca Obtain a copy of the file scalepack stats and place it in the same directory as scale This utility organizes the output of SCALEPACK into a handy format Obtain a copy of runscale and place it in the same directory as scale Runscale is a script file to run SCALEPACK using scale as input and filters output through scalepack stats to display a quick summary of the scaling operation To begin scaling issue the command runscale When scaling is completed a summary of the scaling operation will be displayed The summarized statistics can also be viewed in the file scale stats 1 It is generally better to err on the side of too many preds higher mosaicity than too few lower mosaicity else reflections may be missed Roger Rowlett 29 scalepack lt lt eof scalepack format denzo_ip number of zones 10 estimated error 0 044 0 052 0 065 0 075 0 085 0 11 0 13 0 15 0 18 0 23 error scale factor 1 8 rejection probability 1 e 4 write rejection file 0 5 sigma cutoff 3 0 postrefine 6 reject scale restrain 0 05 b restrain 0 2 ignore overloads Crystal data space group C2 Define images reference film 1 resolution 30 2 2 sector 1 to 280 FILE 1 x hical5 x Control postrefinement fit crystal a 1 to 280 fit crystal b 1 to 280 fitverystal 1 to 280 fit crystal alpha 1 to 280 fit crystal beta 1 to 280 fit crystal gamma 1 to 280 fit Film rotx 1 to 280 fit film roty 1
7. Figure 9 You will need to enter a job title select the appropriate MTZ file to be scaled from your MOSFLM integration define an output MTZ filename different from the input MTZ filename and optionally the Roger Rowlett 35 estimated number of residues in the asymmetric unit The latter is useful if you would like to obtain an estimated average b factor for the data set from a Wilson plot If PNAME XNAME and DNAME were set in MOSFLM before integration these will be successfully read into the job under Define Output Datasets These parameters are mandatory if you are merging two or more datasets together in CCP4i The Scaling Protocol defaults should be fine in 99 of cases w Scala Scale Experimental Intensities _ Customise Scala process default is to refine amp apply scaling Separate anomalous pairs for merging statistics JE Run Truncate to output Wilson plot and SFs after scaling M and output a single MTZ file Ensure unique data amp add FreeR column for 0 05 fraction of data _ Generate Patterson map and do peaksearch to check for pseudo translations Job title scale_highres MTZin Full path momeirrowiett d44n01 HighRes d44n01 highres mtz Browse View _j Override automatic definition of runs to mark discontinuities in data Exclude data resolution less than 5 999 Angstrom or greater than 2 800 Angstrom MTZout d44n01 444n01 highres_scalat mtz Browse View 4 C
8. aliquoted to minimize repeated freeze thaw cycles that are usually deleterious to proteins In general protein solutions should contain the minimum concentrations of buffers salts and preservatives necessary for safe storage In particular the use of high concentrations of glycerol or other polyols in storage solutions should be avoided as this can alter or interfere with crystallization Sample handling Most proteins are sensitive to harsh handling Unless known otherwise proteins should always be maintained on ice when not in the refrigerator cold room or freezer In addition protein solutions should never be subjected to vortexing or vigorous mixing the resulting foaming promotes protein denaturation Before using proteins in crystallization trials it is customary to remove dust and precipitated protein by centrifugation at 14000 xg for 5 10 minutes at 4 C Hanging Drop Crystallization The most common method of protein crystallization is hanging drop vapor diffusion In this method a concentrated protein solution is combined with a solution of a precipitant and allowed to concentrate by evaporation Under the right conditions and with the appropriate precipitant protein crystals will form In hanging drop vapor diffusion a small volume of protein sample and precipitant are combined on a glass coverslip and sealed over a well containing precipitant solution fig 1 Because the precipitant concentration in the mixed drop of protein i
9. and space group number can be found in the log file of truncate Give the file the cel extension File 8 is an example for a C2 crystal space group HS File 8 epmr cel file 232 66 144 73 52 41 90 93 96 90 5 46 X Ray Crystallography Methods Running EPMR EPMR uses an efficient evolutionary search algorithm to find one of many good fits of the search model to the reflection data during each trial The search is repeated for many trials starting with different initial orientations of the search model The results of the best of these trials is assumed to be and often is close to global best fit providing a good model for estimating phase data and constructing the first electron density map The program is customizable by including various switches in the command line some of which are outlined below e o filename sets the stem for the filenames of the output PDB files which will look something like filename 1 best pdb e mn instructs EPMR to place n molecules of the search model into the unit cell The default is to place one molecule in the unit cell e tn instructs EPMR to use the correlation coefficient n as the cutoff value for determining what is a satisfactory molecular replacement solution When placing more than one molecule in the unit cell it is usually desirable to set this value to 1 0 to force an more exhaustive search for the best fit for the first molecule placed This often improves the chance of success
10. cyan Flip_peptide Refi_zone Tor_residue colour_text yellow Dist_define Neighbour_atom Trig_reset Trig_refresh colour_text turquoise gen_symmetry redraw_solv redraw_map next_water skip_5_ca next_ca You may create a custom menu by reading into O an appropriate O database File 11 menu_rowlett pdb is an example of a handy O menu that keeps commonly used commands close at hand and executable at the click of a mouse Commands included in the personal menu will appear in a box on the O display screen The position of this menu is normally controlled by the on_startup file The first line in the file describes this as a menu file MENU composed of text T 40 lines long not counting the first line with a maximum of 24 characters per line The menu can be color coded in blocks by adding colour_text lines the other lines in the file are either existing O commands or references to user script files script files references can be recognized by the preceding symbol Roger Rowlett 59 Some useful script and database files are shown in Files 12 16 A brief description of the files is given in the comment section of the file following the symbols File 17 is a useful O database file that modifies the way O labels residues picked by the mouse displaying the residue type as well as the chain ID e g Arg D160 CA instead of D160 CA File 18 is required when running O in the Windows operating system as described previously
11. edu chemistry xray Some helpful tricks and tips will be given along the way You are encouraged to consult the additional resources for more information Starting and Ending a Linux Session Starting a Linux session To start a Linux session type in your username and password at the welcome screen Do not share your password with anyone else others could potentially make mischief either intentionally or unintentionally in your work area The system administrator will set up an account for you and help you configure your desktop which will look something like Figure 6 w Colgate Protein Crystallography Home Mozilla Firefox Fle Edt View Go Bookmarks Tools Help 5 gt A 4 hnp depanments colgate edu chemistry xray IG Colgate University Department of Chemistry Protein X ray Crystallography Computing Facility Roger S Rowlett Director Home X ray crystallography remains the gold standard for protein structure determination The Colgate University Protein X ray Crystallography Computing Facility provides computational suppor for protein X ray crystallographic analysis and serves to engage undergraduate students in thes critical aspect of biochemistry and structural biology research The latest happenings are found on the page Sample images trom of some of the molecular visualization tools available are shown below Cdrrowiene tabasco shell Konsole Session Edit View Bookmarks Settings Help a H An
12. excellent choices Table 1 Typical Cryoprotecants and Concentrations Required Cryoprotectant Concentration glycerol 30 v v sucrose 30 w v glucose 30 w v ethylene glycol 30 w v MPD 30 v v PEG 400 20000 25 40 v v or w v Once a suitable cryoprotectant solution or solutions have been identified the behavior of protein crystals in these solutions should be observed This is often carried out at the same time as crystal mounting as described below You should observe that the crystal does not disintegrate crack or split during cryo soaking For especially difficult cases you can try sequentially soaking crystals in 15 glucose and then 30 glucose prior to freezing Many 8 X Ray Crystallography Methods proteins otherwise impossible to freeze survive this treatment It is not necessary to soak crystals for extended periods to confer cryoprotection All that is necessary is to replace the solution on the surface of the crystal with the cryoprotectant solution a process that only takes a few seconds of soaking Mounting protein crystals on loops Protein crystals are mounted for diffraction on tiny nylon loops 0 05 1 0 mm in diameter The loops are mounted on hollow rods that are in turn mounted on magnetic caps that are conveniently stored under liquid nitrogen and are easily placed on the goniometer head of the X ray diffractometer A photo of a loop and cap is shown below fig 3 The following
13. far away from the correct solution The purpose of rebuilding is to position the model in a more appropriate starting point for refinement to do its magic Coot is specially designed to integrate well with the CCP4 suite of crystallography programs so it is especially appropriate if you are using MOSFLM SCALA and Refmac One of the many nice features of Coot is the ability to re contour electron density maps on the fly using phase and intensity data written out by Refmac It also has the ability to do very nice real space refinements of segments of the model Most new users of crystallography software will find Coot easier to use and to integrate into the model refinement environment than O Starting Coot and loading a molecule and electron density maps The command for starting Coot is typically aliased to something like coot Usually the command is issued from the local directory from which you are working so that you do not have to specify complete paths to your data files When Coot is started it always asks you if you want to run an auto save file that stores the last saved state Unless you want to start where you left off you can click on No To load a pdb file select File Open Coordinates and choose the appropriate file name The selected molecule will be loaded and displayed as a stick model To open a Refmac generated MTZ file that contains phases and intensities for contourable maps select File Auto Open MTZ and choose the approp
14. for finding a satisfactory solution for multiple placements The default is a correlation coefficient of 0 45 for one molecule or 0 30 for the first of multiple molecules e hn gives EPMR the high resolution limit of data to be used in the search The default value is 4 A Occasionally using slightly higher resolution data can help find a satisfactory solution This value should normally be set to 5A or higher resolution e 1n gives EPMR the low resolution limit of data to be used in the search The default value is 15 A If accurately measured low resolution reflections are available including data out to 25 30A can be useful The general format for invoking the program is epmr o filestem filename cel filename pdb filename epmr where fi Lestem is the stem of the output PDB filename filename cel is the unit cell information file filename pdb is the molecular replacement search model in PDB format and filename epmr is the reflection list file in EPMR format The command line which can be quite long is best put into an executable Linux script file named epmr sh an example of which is shown in File 9 The command can be invoked to run in the background by typing epmr sh amp at the prompt File 9 A typical EPMR executable file epmr m3 t1 0 o 3dimer hica08 cel dimer pdb hica08 epmr gt 3dimer log The script in File 9 will do an exhaustive search correlation coefficient of 1 0 to place 3 molecules of dimer pdb in the
15. group e g P222 P2221 P2 2 2 P2 22 P22 2 P2221 P2212 P2122 Of these space groups only P222 P2 2 2 P2 2 2 and P222 are recognized as standard space groups The others are non standard variants in which the h k I indices have been permuted To convert one of these non standard space groups into a standard one the reflection data indices must be appropriately swapped For example to convert reflection data from P22 2 to the standard P2 2 2 it is necessary to rearrange the indices hkl into klh This is conveniently done in CCP4i e Open a CCP4i session as previously described previously e Select the Reflection Data Utilities module in CCP4i upper left menu bar and click on Reindex Reflections A task window will open Figure 11 e Enter a job name e g reindex and select the appropriate MTZ input filename An output file name will be generated or you can change it to something else e Under the Reindex Details section of the form select entering reflection transformation In this example we have selected h k k l I h to permute the indices hkl to klh Roger Rowlett 39 e Check the box Change spacegroup to and enter the proper standard space group here P21212 e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs m
16. in the X ray beam of a diffractometer and determine if it diffracts to sufficient resolution to justify collecting a full data set The initial crystal screen can often be used to determine the space group of the crystal an important piece of information is planning data collection The exact instructions for collecting data will vary depending on the type of equipment used The following instructions are suitable for using the Unix controlled RAXIS IV detector systems at the National Institutes of Health Figure 4 Figure 4 Source cryosystream and RAXIS IV detector system B at the National Institutes of Health Laboratory of Molecular Biology Bethesda MD Screening crystals for diffraction Preparing the diffractometer The following steps must be carried out prior to commencing data collection e Configure and start the cryostream system to cool and hold your crystals to 95K Depending on the particular cryo system used it can take up to 2 hours for the cryostream to come to temperature e Enter the X ray hutch make sure that the X ray beam is not on and the shutter is not open and immediately verify that the X ray shutter is manually closed Closing the shutter when entering the hutch should become second nature Closing the shutter minimizes the risk of being exposed to direct or backscattered X ray radiation while working in the hutch e Immediately before collecting data energize the X ray source by 1 increasing the
17. log filename should be edited as required To run the command type sca2mt z at the prompt Examine the log file to ensure that the program ran satisfactorily before proceeding File 4 sca2mtz scalepack2mtz hklin hical5 sca hklout hical5a mtz gt sca2mtz log lt lt eof scale ANOMALOUS NO SYMMETRY C2 END eof scale Truncation of reflection data The Linux script t runc File 5 calls the CCP4 program truncate which converts the intensity data in mt z format output by sca2mtz to 42 X Ray Crystallography Methods structure factors In addition truncate also provides useful information about the data set including the average b factor for the data set from a Wilson plot the approximate percentage of the crystal lattice occupied by protein and the scattering anisotropy of the crystal along with other information If you have processed your data in MOSFLM and SCALA as described in the previous section your data has already been converted to structure factors by truncate and this step is unnecessary and you can proceed directly to molecular replacement and or model building and refinement The input file hklin the output file hklout the number of residues in the asymmetric unit nresidues and the log filename should be edited as required To run the script type trunc at the prompt To run this script the CCP4 suite must be enabled by running the appropriate source command which is typically aliased to the command ccp4set
18. molecules typically HOH and attaching a segid typically S It is unlikely that waterpick inp will place all the required water molecules in your structure so it will be necessary to add the rest manually using O as described below Start O and load your protein molecule and electron density maps as usual Issue the water_init command The command will prompt for a molecule name typically SOLV the number of water molecules to reserve space for 100 300 is enough depending on the size of your protein molecule and the resolution of the data set and the number of the first residue for the added water molecules To make life easier later ensure that this number is larger than the number of water molecules added so far You will usually add water molecules in several sittings so you want to ensure you don t accidentally overwrite any existing water molecules To add a water molecule center on an atom close to the electron density you would like to fill and issue the command wat er_add A new water molecule will appear as a little red star superimposed on top of the atom you previously centered on Click on Yes to accept the existence of the new water molecule Turn off the protein molecule so you can see the water molecule more clearly and move the water molecule into the electron density using grab_residue When you are satisfied with the position of the water molecule check it in 3 dimensions click on Yes to accept Note when you pla
19. not all the spots on the image are accurately predicted the mosaicity should be adjusted in the processing parameters pane by clicking on mosaic and changing the value Typical flash frozen crystals have mosaicity values between 0 2 and 0 8 Experiment with various values for the mosaicity until the spots are accurately predicted If you have more spots than predictions increase the mosaicity if you have more predictions than spots decrease the mosaicity You need not be too fine here the mosaicity value will be refined by MOSFLM during integration Good agreement between spots and predictions is a prerequisite for integration of the full data set Integrating the entire data set using MOSFLM Once you have determined the correct parameters for indexing your first image you can refine the unit cell parameters and then integrate a series of frames or a complete data set to catalog all the observed reflections and their intensities In MOSFLM this is conveniently done immediately after autoindexing the first frame of data without closing your MOSFLM session Roger Rowlett 33 If you are integrating a data set from scratch you may want to re run autoindexing before proceeding The following procedure is typical for data set integration It is important to set a few keywords before proceeding to make the data easier to process in SCALA later Click on Keyword input and set the following keywords o PNAME XYZ01 enter a project name
20. o XNAME XYZO1 enter a crystal name o DNAME HighRes enter a dataset name Additional keywords should be entered at this time including information about the resolution range to be processed and the expected separation between spots in mm o Resolution 30 2 8 enter resolution range of reflections to be processed o Separation 0 9 0 9 this keyword is especially useful for data from large unit cells where reflection spacing is tight Lower values allow recognition and quantification of spots closer together Use with care verify that integration boxes are large enough to accommodate entire spots when reducing separation o end Refine unit cell parameters by clicking on Refine cell in the main menu You will be prompted with a series of questions shown below with typical answers Comments are given in italics Give number of segments 2 it is generally best to refine cell parameters with two segments of images Image number for first image of segment 1 1 enter image number Image identifier XYZ enter filename prefix without image number or accept default Use phi values from image header Y Number of images in this segment 4 choose 3 4 images Use the current crystal orientation Y Image number of first segment of segment 2 90 you should choose a second segment that is separated from the first segment by 45 90 in phi Filename for final orientation matrix XYZ highres mat enter filename of your choice Do you want to proc
21. oscillation start 0 00 end 0 50 unit cell 232 649 144 639 52 101 90 000 94 104 90 000 crystal rotx 111 721 roty 23 818 rotz 132 990 END of CRYSTAL ORIENTATION PARAMETERS Fitting Parameters resolution limits 25 0 2 0 space group C2 mosaicity 0 60 oscillation range 0 5 start 0 0 sector 1 to 280 error density 0 6 error systematic 5 0 partiality 0 10 positional 0 070 weak level 5 0 profile fitting radius 25 0 raw data file hical5 osc film output file x hical5 x create directory x prior to run HOxX lt 2 5 2 05 spot radius 0 8 background radius 0 9 overlap spot Refinement Begins start refinement print no profiles fit crystal rotx roty rotz go go go go go go go go go go go go write predictions fit cell go go go go go go go go go go go go go go go fit all fix y scale skew go go go go go go go go go go go go go go go go go go list print profiles 1 1 calculate go end of pack end of job 28 X Ray Crystallography Methods In the zoom window press the nt Box button so that you can observe the spot size and integration boxes Examine the diffraction pattern as it is integrated If everything is working properly the preds should match with the observed spots from frame to frame and there should normally be about as many preds as spots If overlaps red spots exceed 10 of the total the data is likely to be unusable you will have to choose differ
22. protocol is typical Figure 3 Mounting loops and cryovials left Closeup of a 0 50 mm mounting loop right e Obtain and don comfortable Thinsulate gloves to protect your hands from frostbite e Fill a tall dewar with liquid nitrogen and insert and cool a labeled cryo cane to hold your mounted crystal samples Fill a second dewar with liquid nitrogen to periodically top off the first dewar e Obtain a vial clamp and a crystal wand for handling vials and crystal caps Obtain a collection of cryovials fitted with crystal caps with various sizes of mounting loops The caps are color coded to aid in indentification Obtain your crystal tray containing crystals to soak freeze and mount Obtain a spot plate a 20 uL pipettor and pipette tipes and your cryoprotectant solutions Assemble all of these materials around the dissecting microscope Place the crystal tray under the microscope and focus on a well containing suitable crystals Without removing the coverslip determine what size loops are appropriate by holding them under the microscope next to the coverslip You should choose a loop size that is just slightly larger than the crystals e Pipette 10 20 uL of cryoprotectant in a spot plate well Roger Rowlett 9 e Label a cryovial bottom and mount it in the vial clamp e For the next steps you must work quickly as the protein drop may evaporate rapidly causing protein precipitation or crystal cracking e Carefully remov
23. rotate the item about the initially selected atom on the x or y axes by holding down the key and the right mouse button You may rotate the item about the initially selected atom on the z axis byt holding down the fm key and the right and middle mouse buttons When you are satisfied with the orientation of the moved item s select Yes from the menu else select No to cancel the operation Note if you move more than one item only the last item moved will be able to be canceled It is recommended that you normally only move one item at a time and complete that operation before proceeding Alternatively execute a SAVE prior to making complex grab operations so that it is possible to return to a known previous state Grab movements will probably seriously distort the normal bond angles and distances in the main and side chains so the affected region of the model should be regularized before proceeding After regularization described below it may be necessary to use the grab command s again to make minor adjustments Usually 1 2 iterations of moving and regularization are sufficient to correctly place the model into the electron density Regularizing the model The refi_zone command can be used to regularize a manually adjusted model to conform to normal expected bond angles and disitances To regularize a portion of the model refi zone can be selected from the user menu following by 64 X Ray Crystallography Methods clicking on two a
24. section check Combine all input datasets into a single output dataset Change the output dataset name to something descriptive like a11 Optionally enter the estimated number of residues per asymmetric unit to get accurate Wilson Plot statistics including the average estimated b factor for the dataset e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly Examine the scaling statisitics to verify that the combined data set is satisfactory Combined datasets may not have monotonically varying values of Rinerge I o 1 or multiplicity by shell because of discontinuities in the merged data However the merged data should still have overall statistics that conform to what is expected for a usable dataset e The output MTZ file from this procedure is ready for further processing as described in the next section Model Building and Refinement Reindexing Data Sets in CCP4i Sometimes a space group is generally known but the exact space group including screw axes is not immediately known and the data set must be re indexed to conform to standard conventions later For example you may know that a particular crystal is in the primitive orthorhombic space
25. the N or C termini remove them e Leave as is any residues that are identical in both proteins e For mismatches change the molecular replacement residue to Ala except Pro Gly or Ala residues in molecular replacement model should be left as is Gly should be used where Gly appears in the target protein No substitution is necessary for Asn Asp or Gln Glu Phe in the molecular replacement protein is allowed to subsitute for Tyr in the target protein o Val in the molecular replacement protein is allowed to subsitute for Ile in the target protein O O O O The necessary modifications can be easily made using O Coot or Swiss PDB Viewer Note for solving the structure of mutant proteins the ideal search model is an existing solved structure of the wild type protein No modifications need be made to the residues of the molecular replacement model in this case For the purpose of generating an initial electron density map it is probably wise to remove all cofactors e g coenzymes metal ions bound species e g buffers solvents ions and solvent Finding molecular replacment solutions using EPMR Before an electron density map can be generated it is necessary to place the search model molecular replacement protein in the appropriate location of the unit cell There are a number of programs capable of doing this but among the best is EPMR the instructions for which are described here The first task is to convert the truncated mt z f
26. to estimate the most likely number of protein molecules within the asymmetric unit of the unit cell This task can also be carried out in the CCP4i interface by the following steps e Inthe CCP4i main task window select Molecular Replacement from the task menu and click on Phaser A task window will open Figure 13 e Enter a job name e g matthews and the input file name the sorted structure factor MTZ file for the entire dataset 48 X Ray Crystallography Methods e Under Mode for molecular replacement select cell content analysis e Under Composition of the asymmetric unit choose protein and enter the molecular weight of the search model and the number of these molecules you expect in the asymmetric unit If you don t know how many search models are reasonable to enter try 1 e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly aMaximum Likelihood Molecular Replacement Job title matthews Mode for molecular replacement cell content analysis Define data cell content analysis MTZ in y181f23 181f23 unique mtz Browse ju A F F_all SIGF SIGF_all M Resolution range 20 0 A to 2 400 A Composi
27. to rm and remove the correct files with confidence The question mark is a wildcard character for a single character in a filename For example the command rm abcd would remove from the current directory all files exactly five letters long starting with the letters abcd and any other fifth character Brackets are used to enclose ranges of characters allowable in a single character position for selected files For example the command rm mydata 1 0 5 0 9 osc would remove files mydata 100 tomydata 159 if present in the current directory The ampersand amp is used to instruct Linux to run a program in the background In this way you can continue to use the current Linux shell while your program runs Background jobs will continue to run even if you log off the machine For example the command myprog amp would start the program myprog display a PID and return the shell prompt The program will run in the background until it finishes or is terminated with the kill command Roger Rowlett 21 The up arrow key f7 will display the last command typed if your environment is set up appropriately Repeatedly pressing ff will call up additional previous commands Pressing the f key will bring up successively more recent commands Commands that are called up this way can be edited using the f and keys to scroll across the line To execute a command called up and or edited this way press mfe The middle mouse button actuall
28. unit cell described by hica08 cel using hica08 epmr reflection data The best fits for the three placed dimers will be written out as Roger Rowlett 47 3dimer 1 best pdb 3dimer 2 best pdb and 3dimer 3 best pdb The real time output of the program will be sent to the file 3dimer log which can be monitored by using the tail f command EPMR even as efficient as it is will take a substantial amount of time to find a molecular replacement solution for a large unit cell especially if multiple molecules must be placed It is best run as an overnight job Preliminary determination of suitability of the molecular replacement search A decent molecular replacement solution will have an R factor no larger than 0 45 If R gt 0 50 it is unlikely that the molecular replacement solution will be useful If the R factor is satisfactory then the molecules placed in the unit cell by EPMR should be examined for overlaps with themselves or with symmetry generated partners by loading them into Swiss PDB Viewer or O The operation of O is described later If there are no obvious overlaps and the symmetry generated molecules pack well into the unit cell you should proceed else you should re evaluate your molecular replacement solution and perhaps try again using different conditions Preparing EPMR output for use by CNS If you have placed several molecules of a search model into the unit cell they should be consolidated and reformatted before proceeding
29. using only bgroup inp and bindividual inp to re calculate proper b factors CNS or run one cycle of Refmac without ARP_WATERS Then repeat structural validation When all fixable structural anomalies have been addressed you are ready to submit the structure to the Protein Data Bank at http www rcsb org Additional Resources CCP4 http www ccp4 ac uk Crystallography and NMR System http cns csb yale edu v1 1 Coot http www ysbl york ac uk emsley coot doc user manual html The O files http www imsb au dk mok o O for Morons http seqaxp bio caltech edu www hhmi manuals morons o_for morons html Uppsala Software Factory http xray bmc uu se gerard manuals A to Z of O http xray bmc uu se alwyn A Z _ of O A Z_frameset html
30. want to exclude spots close to ice rings N applicable only if you have ice rings Filename for final orientation matrix xyz mat enter a filename of your choice Maximum expected cell edge Angstroms 190 choose a value that you believe is larger than your longest dimension if autoindexing fails you might try autoindexing again with a larger value Do you want to pre refine the solutions N Do you want to proceed Y Select a solution AND a spacegroup from list 10 P41212 A list of possible space groups will be listed along with their penalty functions Normally you should choose the highest symmetry space group that has a low penalty score You should notice a large gap in penalty scores between acceptable and unacceptable space groups MOSFLM will suggest the best solution for you Positional sigma cutoff 2 50 accept the default Do you want to update cell parameters Y Do you want to accept the new beam coordinates Y Do you want to accept this solution Y You should observe a series of red crosses in the graphical window indicating spots that were used to perform the autoindexing Clear these from the image by clicking on Clear spots in the main menu Call up spot predictions by clicking on Predict in the main menu You will observe a series of colored boxes in the graphical window These boxes should correspond to the positions of spots in the image If
31. 4 Bare b nes LINUX ZOO O ee ee 17 Starting and ending a Linux SESSIO E GA OKE AAAA WAN AGA 18 Fil st rage Structure In EMO KC RW OO ORA AREA 18 Commonly used Linux commands s zd O A NE AA 18 Special Linux command line characters and actions useeea aaa aaa aaa aaa aaa zarania cna 20 Input Output redirection za R O O A A ad A dy d 21 Customizing your Linux ENYOMMENK OZ O A R AE 21 Additional TESOUTECS aa ARG AE b A W 22 X ray Diffraction Data Analysis sw a aid asd 23 Indexing the first frame using DENZO awe aa aid Z a A Ak 23 Integrating an entire data set using DENZO e ee esa ea aaa aaa aaa aaa aa aaa aaa aeaa ace 26 Scaling reflection data set using SCALEPACK eeeuao aaa ooo aaa aaa aa aaa aa cana 28 Indexing the first frame using MOSPEM ccc aoc ki dosiada aa kikA 30 Integrating the entire data set using MOSFLM eeeuae aaa aaa aaa aaa wana eaaaaeca ace 32 Scaling reflection data using SCALA aa LR ideal A EA 34 Merging multiple data sets in CO PAB aaa RK AO w AGA 36 Reindexine data sets mi CCPAL AR A RAA AA 38 Additional resourceS eee eee eee een c 39 Model Building and Refinement 2 20000000 00000 000000000000000000000000000000000000000000000000000000000000000 41 Preparing SCALEPACK data for analysis see sseeao see o een aaa ea aaa nawa aewa ace 41 Converting MTZ Tiles to CNS fomat s oai aaa ba 42 Obtaining phases by mol
32. In general the SCALA default settings are very good and scaling of data is quite transparent The following procedure is typical e Configure CCP4 by typing ccp4 setup at the prompt Start CCP4i by issuing the command ccp4i at the prompt The CCP4i graphical interface will open Figure 8 jg CCP4 Program Suite 5 0 2 CCP4lnterface 1 3 20 running on tabasco colgate edu Project d44n01 data Reduction 61s Feb 05 FINISHED mtz2fobs Directories amp ProjectDir Integrate Images 15 Feb 05 FAILED mtz2fobs 15 Feb 05 FINISHED combine View Files from Job _ Da RUR SIATCE Sarai Delete Archive Files Sort Modify Combine MTZ Files Scale and Merge Intensities Kill Job ReRun Job Edit Job Data Preferences Treat Twinned Data 15 Feb 05 FINISHED scalemerge View Any File Import Merged Data 15 Feb 05 FINISHED scalemerge I List of jobs finished or running in this project ___ Mail CCP4 Exit System Administration 7 Figure 8 Main task window for CCP4i Tasks are listed in the left pane jobs in the middle pane and administration functions in the right pane j e If you have not already done so set up and select a project directory by clicking on Directories amp ProjectDir in the administration pane e Select the Data Reduction module in CCP4i upper left menu bar and click on Scale and Merge Intensities A task window will open
33. PROTEIN X RAY CRYSTALLOGRAPHY METHODS pa y 3RD EDITION Roger S Rowlett Department of Chemistry Colgate University 2006 Table of Contents Tntrod tti h kon R Ed 1 ADV VATIONS PEA wini wadi adi akc di ide 1 Crystallization of Pro fcins jcc pccisssscnsccvsceescsosecesacouvscecssuesccvessasenceuvensavcuaudeorecdedenneueensaseunustanencdeoeses 3 Preparation of protein samples a wio ashes iG eas ee cette nurs cela oa tana Gel 3 Hanging drop crystallization Ga ARA sd EG a AA 3 Initial screening of crystallization conditionS 2 e e esa awa awwa 4 Optimizing crystallization conditions waz dA RAN A R O 5 Additional TESOUTCE ss dA A O EA tase 6 Soaking Mounting and Freezing Protein Crystals 20s2 000000000000000000000000000000000000000000000000 7 Screening for a suitable cryoprotectant ccceccceessecetecceeceeeceeeeeeseecueceeeeeeeeeaeecsueeneenaes 7 Mounting protein crystals on loops saa i ai da wia kad 8 S akino 1n OAS a oto ki AE W AW i Akk 9 Additional FESOULCES lt w WPA RO ZA kW 9 X Ray Diffraction Data Collection 00 2 00 000000000000000000000000000000000000o0oooooo oo ooo oo ooo oo 0oo00000000 11 Preparing the diffractometer for data collection e e eo aaa aaa aaa aaa aaa 11 Screening crystals for Ci MTAC MOM aa ad ni TRA GA AO BRA KO eames 11 Collecting a complete diffraction data Set aaa wska O WA dw 14 Shutdown procedure ws aaa di O a ka a W kia td 1
34. a44 which would center the screen on the a carbon of residue 44 in chain A of the molecule The graphical viewing enivronment of O is shown in Figure 17 Roger Rowlett 61 w O Linux 5 X Object_menu 2FOFC_4 FOFC _1 Dial Menu Rot Rot Y user menu is on the right Inspecting a molecule in O The molecule can be manipulated on the screen with the mouse Press the right mouse button and drag to spin the molecule To zoom hold the middle mouse button while scrolling up and down To slab cut away the molecule hold the middle button while scrolling left and right Pointing at an atom and clicking will display an identifying label You may turn on or off the display of various objects in the model by clicking on the appropriate name in the object menu Generating symmetry atoms It is frequently useful to generate symmetry related atoms in the displayed model in order to observe interactions at protein protein interfaces or to get a more accurate view of an interfacial active site etc O must be initialized with the sym_setup command prior to generating symmetry atoms The sym_setup command will prompt for the molecule name unit cell dimensions and space group If there is a CRYSTAL record in your input PDB file the correct default values will be presented otherwise you will have to enter them manually Sym_setup need only be executed once To generate symmetry related atoms around the currently selected atom issue t
35. and all commands you type will normally apply to the files in this your local directory unless instructed otherwise Your local directory might be something like home jdoe That is unless instructed otherwise all files will be read and written to the jdoe directory of the home directory of the machine you are logged in You can find out where you currently are by typing the pwd command you can make a new directory in the current directory with the mkdir command or you can change your local directory to another with the cd command These and other commands are described below The string home jdoe filename describes an absolute path to the file filename a complete set of instructions to locate the file in question The leading slash indicates that this is a complete path starting with the directory home The string datafiles filename is a relative path which describes how to locate a file from the local directory A relative path does not start with a leading slash For example if you were currently in the directory home jdoe the relative path datafiles filename would point to the absolute location home jdoe datafiles filename Relative paths can save a lot of time when typing commands It is important for new Linux users to know that Linux will not generally protect you from yourself For example deleting files or directories in Linux is absolutely positively no turning back irretrievably FINAL You cannot recover files you delete ac
36. another easy Coot task From the Model Fit Refine task menu select Add Terminal Residue and click on the terminus of the molecule you would like to add to Coot will add an alanine residue and make its best guess of the appropriate conformation You may have to mutate the added residue to the correct side chain and adjust its conformation to match the observed electron density Adding Water Molecules to a Model using CCP4 and Coot Adding water molecules to a structure is most easily accomplished by running Refmac together with ARP_WATERS to automatically and iteratively place water molecules in the structure according to specified and unbiased electron density constraints To enable ARP WATERS select the Cycle with arp_waters option in Refmac Figure 19 Under Refinement Parameters select 10 20 cycles of ARP_WATERS Careful selection of the ARP_waters parameters is necessary for successful automated placement of water molecules In particular e The maximum number of new waters to be found per cycle should normally not exceed 0 08 x N R where N is the total number of protein atoms in the structure and R is the resolution in angstroms Therefore for a 2 3 A structure with 3000 protein atoms the number of new solvent atoms found each cycle should not exceed 20 e The threshold electron density for water addition is typically set to 3 46 e The number of waters to be removed each cycle should normally be set to 25 100 of the number o
37. ata set in preparation for producing an electron density map you should examine the x values In a properly scaled data set the overall y and the X for the individual data shells should be 1 00 0 02 The x can be adjusted for each shell as follows o Ifall x values are too high adjust by raising the value of error scale factor Ifally values are too low adjust by lowering the value of error scale factor o To adjust x in individual resolution bins change the value of estimated error for that bin If y is too high raise the value of estimated error If X is too low lower the value of estimated error o Rescale data adjusting error scale factory and or estimated error until all resolution bins have a x value of 1 00 0 02 and the overall 4 is close to 1 0 The output reflection sca file is the raw data from which the data will be solved If this file is satisfactory the image files may be compressed backed up and removed from computer to save space Indexing the first frame in MOSFLM When screening crystals for diffraction or before analyzing an entire data set it is necessary to index the reflections observed in the first frame in order to obtain the exact orientation of the crystal with respect to the X ray beam and to make a preliminary determination of the space group and unit cell dimensions The program MOSFLM which is part of the CCP4 suite of protein crystallography programs can be used to perfo
38. atch of the other dataset It is simplest to add a multiple of 1000 to the original batch number e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly e Open a new Sort Modify Combine MTZ files task window Enter a job name e g combine and enter the MTZ filename of the renumbered MTZ file from the previous steps Click on Add File and enter the MTZ filename of the scaled intensities corresponding to the dataset you wish to combine it with Finally select an output MTZ filename 38 X Ray Crystallography Methods e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly e To scale and merge the sorted files open a Scale and Merge Intensities task window e Enter a job name e g merge and select as your input MTZ file the sorted and combined MTZ file created in the previous job Select and output MTZ filename and in the Define Output Datasets
39. ce a new water molecule all others will be erased Immediately after adding a water molecule redraw the solvent molecule using the menu item redraw_solv found in the user menu loaded with menu_rowlett odb This will usually discourage you from filling up the same electron density with more than one water molecule Navigate and repeat adding water molecules using water_add as required During subsequent rounds of refinement with added water molecules you may find it necessary to add additional water molecules or remove them Water molecules with poor occupancy and high b factors typically have electron density that shrinks like a prune during repeated rounds of refinement Water molecules with tiny electron density surrounding them and or b factors in excess of 50 are candidates for removal from the final model Visualizing Molecules and Electron Density Maps in Coot Coot is a very powerful and easy to use molecular visualization and model building program written by Paul Emsley University of York England Using Coot it is possible to visualize the quality of fit of models to electron density data and also to interactively alter the 66 X Ray Crystallography Methods model to better fit the data The latter of these activities termed rebuilding is essential to the refinement process Although refinement programs are very sophisticated it is not now possible for an automated refinement to find the best fit of model to data if the model is too
40. cept or reject the fit which will be highlighted in the graphics window For more precise control over side chain fitting Edit Chi Angles should be selected Click on the side chain of the desired residue and alter individual chi angles by sliding the mouse back and forth on the graphics screen The new conformation will be highlighted To quickly shift between chi angles you can use the number keys pressing selects the first chi angle 2 selects the second etc To complete the operation select Accept or Cancel in the dialog box Adjusting main chain conformation If you have to adjust the main chain trace it is unlikely automated methods will work else it would have been fixed already Typically the best way to adjust main chain conformation is by moving individual atoms and then regularizing the final result To move atoms in the structure select Rotate Translate Zone from the Model Fit Refine task menu Click on any atom in the residue you wish to move To move an entire residue simply click and drag To move a single atom click and drag Coot will automatically make and break atomic connections according to interatomic distance so proceed cautiously to maintain the correct main and side chain connectivity Select OK to accept changes or Cancel to abandon Regularizing the model It is often necessary to clean up manual adjustments by regularizing the structure so that it conforms to normal bond angles and lengths This is esp
41. cidentally Therefore proceed with care and caution when cleaning up data A list of commonly used Linux commands follows in the next section Commonly Used Linux Commands The following is an alphabetical list of a common Unix commands that you might use for routine crystallography work and file maintenance Please note that Linux commands unlike DOS commands are case sensitive So PWD is not the same as Pwd as pwd Filenames are also case sensitive most users avoid using capitalized text in filenames for ease of typing and to prevent confusion e cd directory change your local directory to a new location If you issue cd with no argument it will take you to your home directory Roger Rowlett 19 e chmod permission filename change permissions for files You must provide a filename and one or more permission arguments with this command The permission argument includes an optional group user group others all and a permission execute read write For example to allow a file to be executable you would type chmod x filename To make a file readable and executable by all users but not writable you would type chmod a rx w filename This command is most often used to make scripts you write executable which is not the default For example chmod x filename would make a file executable for the user in addition to whatever permissions it already had e cp filename filename2 copies a file from one location to another For exa
42. ction This first edition of this manual was originally written in May 2003 to provide a compendium of up to date commonly used methods routinely used in the X ray crystallography laboratory in the Molecular Structure Section of the Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health Much of the information contained in this manual was gleaned from my knowledgeable kind and very clever colleagues in the Laboratory of Dr David Davies Special thanks go to Drs Thang Chiu and Jessica Bell my very patient tutors I am forever in debt to their mentorship The second edition was completed in May 2005 and incorporated additional material related to the conduct of protein X ray crystallography as carried out in my undergraduate research laboratory at Colgate I am indebted to my first crystallography research students Ariel Herman 04 and Joey Lee 04 for their patience with the first edition Their laboratory experience inspired improvements and updates to the second edition The third edition was completed in June 2006 and incorporates entirely new sections on using CCP4i Refmac Coot and Phaser With Coot the CCP4 suite has become more accessible and easy to use than ever for undergraduates My colleague Gino Cingolani deserves credit for convincing me to try Coot and Refmac in the undergraduate research environment Many thanks we like it And my collea
43. d the resolution range of the reflection data to be used typically all of the data the type of map u 2 for 2F F maps and u 1 for F F maps and the name of the output map file typically 2fofc map or fofc map The electron density maps are very large and should be immediately converted into O maps which are about 1 10 the size of the CNS maps After the conversion the large and now unnecessary map files should be deleted To do this type the command map_to_omap map at the prompt This will invoke the utility MAPMAN and do the necessary conversions If the conversion fails due to lack of memory common for large maps you will need to set the environment variable MAPSIZE to a larger value Choose a value slightly larger than that shown in the MAPMAN error message e g setenv MAPSIZE 10000000 Note that the environment variable MAPSIZE is all capitalized The resulting model and maps should be examined in O described later to see if they are usable Examine the display to see if much of the model is contained within the 2F F map In addition examine the F F map for key areas of positive density e g known metal ions cofactors or bound inhibitors or substrates If you observe positive density in the correct areas of the model chances are very good your molecular replacement solution is usable and you should proceed with refinement Further model refinement using CNS After the initial rigid body refinement the mo
44. del is typically subjected to simulating annealing which essentially shakes up the model in a random way followed by slow cooling to find a better less model biased fit to the experimental data After this step the model is typically taken through repeated rounds of whole molecule minimization b factor refinement and the generation of new electron density maps for visualization in O At the end of each refinement round manual adjustments are made to the main and side chains to bring them into better compliance with the electron density map At the end of the first round of refinement residues in the molecular replacement model should be changed to the proper side chain in the target molecule and oriented properly in the electron density As the refinement proceeds it may become obvious that some portions of the protein notably the N and C termini are not visible in the electron density map and should be removed Alternatively as the refinement proceeds new regions of electron density may become obvious allowing the addition of residues to the model especially at the N and C termini When the R factor has dropped to 0 30 it is appropriate to begin adding cofactors and other bound species that are clearly delineated by electron density Finally as R approaches 0 24 or has reached the point that no further improvement is possible clearly delineated water Roger Rowlett 53 molecules can be added Typical proteins will reac
45. e Show Symmetry Atoms box Many protein crystals display non crystallographic symmetry and this can often be used to advantage in the early rounds of refinement to increase signal to noise Coot will automatically find non crystallographic symmetry in your molecule and display overlay traces of symmetric protein chains upon request To display non crystallographic symmetry choose Roger Rowlett 67 Edit Bond Parameters and tick Yes in the Draw Non Crystallographic Ghosts box Non crystallographic symmetry traces will be overlayed on the A chain of your protein gt v ek 5 x File Edit Calculate Draw Display Manager Measures Validate HID Reset View Help mol no 0 ZN 144 230 ZN occ 1 00 bf 21 ele ZN pos 6 32 68 65 11 05 Figure 18 The Coot graphics window Recovering a session after a program crash Coot is a work in progress and has been known to crash unexpectedly Fortunately Coot is pretty good at saving your work as you go along minimizing the chance that you will lose your work To recover from a program crash up to but not including the last program edit open Coot read in the pdb you were last working on and select File Recover Session from the menu After your PDB file has been updated you may read in your electron density maps and resume 68 X Ray Crystallography Methods Basic Model Building Tasks in Coot Common tasks in rebuilding models to better fit the electron density maps a
46. e as cryoprotection if desirable and practical Typically the concentration of ligand used should be 10 1000x the dissociation constant K4 if it is known Soaking for 10 30 min should be sufficient to populate the protein in the crystal if the binding site is accessible in the crystal lattice If protein molecules pack in the crystal in such a way as to obscure the ligand binding site or if crystals do not tolerate extended soaking without cracking or dissolving then co crystallization with ligand should be attempted Additional resources e Hampton Research http www hamptonresearch com e Flash Cooling A Practical Guide http www rose brandeis edu PRLab Crystalizations cool Each crystal should be labeled with a unique identifier so that it can be specifically identified later for diffraction screening and data collection For example Human carbonic anhydrase II crystals might be labeled HCAII 01 HCAII 02 etc Cryocanes can be labeled with the first of a sequence of vial names contained within them for easy location in the storage dewar Drop evaporation will be especially problematic during the winter months when indoor humidity levels are very low Working at 4 C may minimize this problem 10 X Ray Crystallography Methods Roger Rowlett 11 X ray Diffraction Data Collection Before a data set can be collected from which the final structure of the protein can be deduced it is necessary to mount a crystal
47. e it under a microscope and examine each of the drops for protein precipitation or foreign objects glass shards fibers plastic bits and make a notation of any drops that are not clear Place the plate in a quiet place at the appropriate temperature and leave it undisturbed for at least 24 hours Figure 2 A Hampton Research 24 well VDX plate and siliconized coverslips Initial Screening of Crystallization Conditions Strategy The determination of promising protein crystallization conditions is typically done using a sparse matrix screen in which a protein is subjected to widely varying pH salts and Up to 40 uL of protein can be used if desired Protein concentrations from 5 20 mg mL are typical a protein concentration of 10 mg mL is a good starting point for initial screens Roger Rowlett 5 precipitants There are excellent commercial screening kits available making it generally unnecessary to mix your own initial screening reagents The following commercial screens are recommended in the order that they should be employed 1 Hampton Research Crystal Screen This screen contains 50 reagents Screen conditions 25 and 27 have a historically poor record of producing crystals and these two can be omitted in order to conduct the screen in two 24 well plates 2 Hampton Research Crystal Screen 2 An extension of the original Hampton Research sparse matrix screen Two conditions can be omitted for conducting a two plate scre
48. e mismatched residues in the search model to conform with that of the target molecule Before performing this task the command mut__set up must be issued to initialize O for this task Once mut__set up has been successfully executed a residue is mutated by issuing the mut_replace command You will be prompted separately for a molecule name and the name of a residue to change and what to change it to If desired you can do this all at once on the command line e g mut_replace hica a181 phe would change residue 181 in chain A of the molecule hica to a Phe residue You will continue to be prompted for additional mutations until you type a blank return at the prompt Once the mut replace command has been successfully completed the entire molecule will disappear from the screen Redraw the molecule with the zo end command The mutated residue s will display in purple At this point it is useful to use the Lego_side_chain or tor_residue commands to adjust the new side chain into its electron density This task is described below Adjusting side chain conformation Two approaches are available for manipulating side chain conformation The lego_side_chain command allows the user to choose from among a population of commonly represented conformers of a particular side chain Sometimes this is all that is necessary to achieve a fit close enough for minimization For finer control of side chain conformation the tor_residue command is preferred as this wi
49. e the coverslip with the desired crystals and place it drop side up over an empty well of the spot plate e Mount an appropriate size loop on the crystal wand and fish out a crystal The loop should be just larger than the crystal If you maneuver the crystal close to the edge of the drop it will be easier to pick up e Place the crystal into the cryoprotectant solution by touching the loop to the drop e Observe the crystal under the microscope to check for cracking or disintegration It is not necessary to soak the crystal for more than a few seconds in order to confer cryoprotection If there are no problems fish out the crystal in the loop and immediately plunge it into liquid nitrogen and keep it there If the crystals crack or disintegrate you need to find another cryo soak e Immerse the empty cryovial into the liquid nitrogen until it stops bubbling Keep both the crystal cap and the vial under the surface and screw the crystal cap into the cryovial and mount the vial in the cane e Mount additional crystals as required before the drop evaporates or you run out of crystals e Store frozen cryovials in a liquid nitrogen storage dewar for future use Soaking in ligands Occasionally it is desirable to determine a protein structure in the presence of a bound small molecule One method of preparing such protein ligand complexes is to soak a crystal in artificial mother liquor containing an excess of ligand this can be done at the same tim
50. ecially helpful when adjusting the main chain To do this select Regularize Zone from the task menu Click on two atoms in the structure between which the structure will be regularized typically plus and minus at least one residue from the area in which manual changes were made so that changes can be blended into the overall main chain trace Roger Rowlett 69 Writing out a PDB file To save your structural edits select File Save Coordinates from the main menu You will be prompted for the molecule to save several may be open in the graphics window at the same time and be expected to select a filename Give the filename a pdb extension to help identify it for later use Adding Ligands and Cofactors using Coot Adding ligands to a protein Adding ligands and cofactors is ridiculously easy in Coot From the main menu select File Get Monomer and enter the three letter code of the desired ligand cofactor or metal A complete list of monomers can be found in the CCP4 documentation The selected molecule will be placed at the center of the display Move the ligand to the desired location using Rotate Translate Zone in the Model Fit Refine task menu The coordinates for the cofactor can be written out as a separate PDB file for manual merging into the protein coordinate file or the coordinate can be appended to the end of any displayed PDB file by selecting Calculate Merge Molecules Adding additional amino acids to the structure This is
51. ecular replacement e seeua eau aaa aaa aaa aaa aaa ca aan 43 Constructing a molecular replacement model ee seuae ea aaa aaa aaa aaa aaa enia 44 Preparing the first electron density map LA o A 50 Finding molecular replacement solutions using EPMR e e eeeoeaa aaa aaa aaa awa 44 Finding molecular replacement solutions using Phaser ee e eee asaa aaa naci anwa 47 Initial model refinement using CNS awa GR eds R 50 Further model refinement using CN add O Od i dd 52 Refining structures using CCPA sia woda yi A aa 54 Visualizing molecules and electron density maps in O eea ae eaa aaa aaa aaa aaa ca aaca 48 Basic model building tasks in O ae Ob boki Ga 62 Adding ligands and cofactors to a model in O sssessssesssssesseessesessseessesersseesseserssressessess 64 Adding water molecules to a model using CNS and O sssssssnsssssssssessssessesrssssessessrssressessees 65 Visualizing molecules and electron density maps in Coot s sessessssessesessseesseserssressessees 65 Basic model building tasks in Cb ada bO W A A O AA wh 68 Adding ligands and cofactors using Coot e seeee eee a ea aaa aaa aa aaa aaa aware caaacca 69 Adding water molecules to a model using CCP4 and Coot se seuaoauaaa aaa acaaaeea 69 Valhd ting SUCCESS OT 71 Additional resources ici wd cence Seco elas Raw WAGA CA Gad ss Sada PAG ee cen 71 Introdu
52. ediately switch the shutter from EXTERNAL to CLOSED e Return the X ray source to minimum power by 1 turning the current down to the minimum level and then 1 turning the voltage down to the minimum level Roger Rowlett 15 e Move the cryostream head back to allow sufficient clearance for removing crystals from the goniometer without touching the X ray source beam stop or cryostream head e Unlock the axis of the goniometer and swing it to an angle where the notch on the crystal cap will line up with the notch on the cryotongs when they are used to remove to crystal e Clamp the empty cryovial in the vial clamp and set it aside e Insert the cryo tongs into the liquid nitrogen and hold them there until all bubbling stops This may take 30 60 seconds The tongs will cool faster if they are held open during cooling It is important that the cryo tongs are fully cooled before proceeding e Quickly remove the cryo tongs and remove the crystal cap from the goniometer The tongs should be oriented so that when they are opened to grab the cap the crysostream can blow into the opening between the two halves of the tongs This will prevent ice ring formation or crystal melting e Immediately plunge the cryotongs and attached crystal cap into liquid nitrogen and leave it submerged e Immerse the crystal wand into the liquid nitrogen until it stops bubbling vigorously Use the crystal wand to remove the crystal cap from the cryotongs Keep the cry
53. eed Y cell refinement proceeds this will take a few minutes Reset missets to those of the first image Y When cell refinement is completed turn on predictions by clicking on Predict in the main menu Spots should be accurately predicted Integration can be commenced by clicking on Integrate in the main menu If desired additional keywords may be entered via the Keyword input item on the main menu before proceeding You will be prompted with a series of questions shown below with typical answers Comments are given in italics Do you want to update any of these N not necessary unless analyzing synchrotron data 34 X Ray Crystallography Methods Give first last image numbers 1 166 enter image data range Use phi values from image header Y Give BLOCK and or ADD keywords if required press g Refine cell parameters N Write a new MTZ file for each block of data N MTZ filename XYZ highres mtz enter filename of your choice with mtz extension Do you want to proceed Y e Exit the MOSFLM graphics window by clicking on Save Exit in the main menu Do not close MOSFLM by clicking on the upper right hand corner of the MOSFLM graphics window The program will crash e Exit MOSFLM by typing exit at the MOSFLM prompt Scaling reflection data using SCALA If integration has gone well you can proceed to scaling data using SCALA The most convenient way to use SCALA is through the CCP4i interface
54. electron density map L 0625596492496 aeo E 551pm Figure 6 Typical Linux session using KDE desktop on a workstation in the Colgate Protein X ray Crystallography Computing Facility The Firefox browser and a terminal window are open on the desktop in this figure 18 X Ray Crystallography Methods Starting a Linux Shell Most crystallography programs and utilities run from a shell window which is basically a text window into which you can type Linux commands To open a new shell in Linux click on the terminal icon in the toobar 7 from the left in Figure 6 A new window should open with a command prompt such as ancho In this case ancho indicates the computer to which you are logged in and is the shell prompt Commands that you type will appear after the prompt symbol Shell windows can be closed by typing exit at the prompt or by clicking the upper right corner of the window Ending a Linux session To end a Linux session not to be confused with exit froma shell window right click on the desktop and select Logout Always log out of your session when you are away from your terminal for more than a few minutes File Storage Structure in Unix Linux File directory structures are similar to that of DOS the precursor to Windows Indeed DOS now called the Command Prompt in Windows is a derivative of Unix and shares many common commands and functions When you start a Linux session you will be located in your home directory
55. em to something else e The crystal project and dataset names should be automatically recognized from your input file e The default number of refinement cycles 10 is usually adequate 56 X Ray Crystallography Methods bgRun Refmac5 Initial parameters from home rrowlett y181f2 3joldfiles CCP4_DATABASE 30_refmac x Job title refmac Do restrained refinement using no prior phase information input _ Input fixed TLS parameters Cycle with arp_waters to analyse solvent model uses old version of ARPfwARP Generate weighted difference maps filesin CCP4 format MTZin y181f23 yi 81f23 3 mtz FP F_all Sigma co it i WJ O PDBin y181f23 morze 000 erowse wew PDB out yisizs yiei sapae 0000000007 owe ww a Create harvest file in project harvesting directory Harvest project name y1 81f23 and dataset name fan Do fio cycles of maximum likelihood restrained refinement Use hydrogen atoms use if present in file ana _j output to coordinate file _ Resolution range from minimum 18 309 to 2 400 Use weighting term 0 04 JE Use experimental sigmas to weight Xray terms l Refine isotropic temperature factors M Exclude data with freeR label FreeR_flag with value of jo Use Babinet scaling Bulk solvent scaling for resolution range i 9 909 to 2 400 Detennine scaling using the working set of reflections M Use experimental sigmas W Calculate the contributi
56. en Evaluating screens Plates should be examined under the microscope and evaluated for protein crystallization after 24 hours and every day thereafter for a week After one week plates should be examined weekly Record in your notebook results for each drop Suggested categories and abbreviations to use are as follows with comments e Clear drop C no changes in drop e Precipitate P typically light brown and granular Crystals sometimes form from such precipitates If the precipitate is thick and swirly this is very bad and unlikely to form crystals e Precipitate phase separation PP typically light brown and granular with little blobs that look like oil drops Crystals sometimes from at the edge of phase separations e Microcrystals MX Difficult to distinguish from precipitate however unlike precipitated protein microcrystals have a shiny appearance Optimization of conditions may result in larger crystals e Needle cluster NX Beautiful but often useless for crystallography Optimization of conditions may result in less needle like forms e Plates PX if not too thin may be useful for crystallography Optimization may result in better crystal form Plate clusters may be separated in to suitable single crystals e Rod clusters RX If separable into single crystals may be useful for crystallography e Single crystals X the Holy Grail large individual crystals with blocky dimensions Optimizing crystal
57. ent integration conditions or change your data collection parameters to generate better separated reflections Examine the integrate out file as it streams out In particular o Examine the mosaicity histogram for each frame If the mosaicity is properly set most of the bins should be filled and should decrease smoothly as you read down the screen If the histogram is jagged or non monotonic there is a problem with the mosaicity setting or worse o Examine the x for the fit on each frame Ideally it should be near a value of 1 for x y and partials Values gt 4 are cause for concern If all has gone well you can proceed to data scaling as described in the next section Scaling Reflection Data using SCALEPACK Before integrated reflection data can be used to produce an electron density map it must be scaled and evaluated for completeness and quality Scaling is done by a stand alone program SCALEPACK The following procedure is typical for scaling data Obtain and edit the file scale file 3 which contains SCALEPACK instructions for scaling your reflection data You should o Setthe estimated error for each of the bins to 0 05 o Setthe error scale multiplier to 2 0 o Choose the frames you would like to scale in the sector line and all of the fit and add partial lines o Choose the resolution range of the scaled data in the resolution line o Edit the filename for the appropriate reflection files on the file line o
58. enu in the administration functions pane of the CCP4i window to verify that the job has run correctly JReindex Reflections Job title reindex MTzin y181f23 181f23 0 mtz Browse vew MTZout y181f23 181123 0_reindex1 mtz _ Browse vew Define transformation matrix by entering reflection transfonnation Use matrixh q kel I h M Change spacegroup to P21 212 Reduce reflections to the asymmetric unit Figure 11 Reindex Reflections task window Mandatory fields are highlighted in color Additional Resources e HKL Research Inc http www hkl xray com e CCP4 http www ccp4 ac uk 40 X Ray Crystallography Methods Roger Rowlett 41 Model Building and Refinement Obtaining a structure from your data is an iterative process that requires supplying a model of your protein structure comparing it to the electron density map derived from the data and model and rebuilding the model When the model and electron density map are in sufficient agreement the model is regarded as a good approximation of the protein structure The most difficult problem in this modeling process is obtaining information about the phase of the observed reflections The intensities are accurately measured in your experimental data set In order to produce accurate electron density maps it is essential to have both accurate intensity and phase information Approximate phases can be obtained by collecting additiona
59. er Rowlett title DENZO autoindexing Detector Information format raxis4 100 use beam wavelength 1 5418 error density 0 6 error systematic 5 0 partiality 0 15 positional 0 050 weak level 5 0 film rotation 180 0 x beam 150 2 y beam 151 6 y scale 1 0 skew 0 000 cassette rotx 0 roty 0 profile fitting radius 25 0 resolution limits 30 0 2 0 distance 180 0 Fitting Information mosaicity 0 7 raw data file hicalS osc space group C2 oscillation range 0 5 start 0 0 sector 1 Hoz print 2 5 215 spot radius 0 8 background radius 0 9 overlap spot Refinement fix x beam y beam fit cell fit crystal rotx roty rotz go go go go fit all fix y scale skew fix x beam y beam go go go go go go go go go go go go go go go go go go go go go go go go go go go go write predictions print statistics peak search file peaks file go go go go go go go go go go go go go go go go go go go go go go go go go go go go write predictions go go go go go go go go go go go go go go go go go go go go go fit all go go go go go go go go go go go go go go go go go go go go go list 25 26 X Ray Crystallography Methods Integrating the entire data set using DENZO Once you have determined the correct parameters for indexing your first frame including the space group you can integrate a series of frames or a complete data set to catalog all of the observed reflections The programs XDISP and DENZO are used t
60. er appropriate software for indexing and preliminary assignment of space group as described in the section on X Ray Diffraction Data Analysis e Perform the appropriate shutdown procedures vide infra Figure 5 A crystal properly mounted and centered on the goniometer head and cooled by the cryoststream 14 X Ray Crystallography Methods Collecting a Complete Diffraction Data Set The physical steps for collecting full data sets are nearly identical to that of collecting screen data e If your crystal is not already mounted and aligned follow the instructions for mounting crystals as described in the previous section e Swing the goniometer to 0 or other desired starting angle and lock the goniometer head e Remove all your tools and dewars from the hutch You will not be able to retrieve them during data collection e Switch the X ray shutter from CLOSED to EXTERNAL exit the hutch and close the door e Start the R AXIS data collection software if it is not running already by entering the Unix command start amp at the prompt e Enter your data collection parameters For data collection is is typical to collect data over a total rotation range of 45 180 The camera distance should be set close enough to measure the highest resolution spots observable but far enough away to resolve the closest spots in the diffraction patterns For maximum efficienty in data collection the oscillation range should be set to the maximum val
61. f water molecules to be found e The threshold electron density for water removal is typically set to lo e The water chain in the protein is typically labled W or S e The remaining CCP41 ARP _ WATERS defaults are acceptable 8 A description of CCP4 recognized monomers can be found at http www ccp4 ac uk html lib_list htm For example the zinc ion is ZN bicarbonate ion is BCT sulfate is SO4 phosphate is PO4 etc 70 X Ray Crystallography Methods bgRun Refmac5 Initial parameters from home rrowlett y181f23 0Idfiles CCP4_DATABASE 33_refmac5 def BEE Job title jadd_waters Do restrained refinement using no prior phase information input _ Input fixed TLS parameters M Cycle with arp_waters to analyse solvent model uses old version of ARP WARP Generate weighted difference maps files in CCP4 format MTZin y181f23 yi 81f23 3 mtz Browse v az FP F_all Sigma SIGF_all MTZout y181f23 y181f23 4cjunkmz Browse View m PDBin y181f23 oma Browse View PDBout y181f23 y181f23 4cunk pdb Browse view Library y181f23 ytetf23 zt lbcif Browse View zi Data Harvesting Refinement Parameters Do ro cycles of maximum likelihood restrained refinement in each Refmac run and 1 cycie s of ARP_waters Use hydrogen atoms use if present in file _ ana _ output to coordinate file _ Resolution range from minimum 19 909 to 2 400 Use weighting term 0 04 W Use experimental si
62. free presumably because of slight model bias but generally these values are no more than 5 0 05 apart If R free R test gt 0 05 you should investigate further or take steps to reduce model bias such as simulated annealing Calculating electron density maps You should calculate two types of electron density maps for modeling purposes A 2F F map is calculated from 2 x the observed minus the calculated electron density This map resembles the electron density of the target molecule and should largely define the main and side chains of the model A F F map is calculated from the 52 X Ray Crystallography Methods difference of the observed and calculated electron densities This map is useful for identifying region of electron density not explained by the model Positive F F density indicates that there is electron density present not explained by the model e g a missing cofactor solvent buffer or ion molecule or a misplaced or missing main or side chain Negative F F density indicates that there is less electron density in the data than is predicted by the model e g a misplaced main or side chain Electron density maps are generated by the CNS module model_map inp Experienced users usually save two separate CNS scripts named 2fofc inp and fofc inp to save time generating maps Required inputs are the pdb mt f and cv files the unit cell parameters and space group any extra top or par files require
63. gand pdb can be combined using a text editor for further refinement or model building Adding additional amino acids to the N or C terminus You may discover during refinement that you can see additional electron density beyond the termini of your search model which may not include the entire gene coding sequence Additional amino acid residues can be added much as are ligands except that excellent pdb files for additional residues can be Roger Rowlett 65 obtained by extracting and copying appropriate portions of the protein molecule using a text editor These protein fragments can be repositioned using the grab_residue command and the extra amino acids can be written out to a separate pdb file To incorporate these extra amino acids into the model renumber the residues appropriately in MOLEMAN2 and combine with the original protein pdb file using a text editor It might be useful to examine the combined model in O and perform a refi_zone to clean up the geometry of the junction between the original protein and the extended residues Adding water molecules to a model using CNS and O The first batch of water molecules can be added automatically by using the CNS script waterpick inp This will append coordinates to the end of your pdb file that correspond to appropriate electron density found to match reasonable geometric constraints for hydrogen bonding to the protein molecule The script includes instructions for naming water
64. gmas to weight Xray terms W Refine isotropic temperature factors jm Exclude data with freeR label FreeR_flag with valueoff0 Sety Geometric Restraints Setup Non Crystatiographic Symmetry NCS Restraints ARP_waters Parameters Remove 25 solvent atoms from density below 1 0 sigma W Merge solvent atoms closer than Ez New to old solvent atom distance greater than 2 2 less than 33 Min distance between new atoms rza Find cw new solvent atoms in density above ao sigma and assign to chain wo Dala Output to MTZ file Scaling Maximum Likelihood Parameters Monitoring Geometric parameters Developers Options Save or Restore Figure 19 Refmac task window with ARP_WATERS option enabled Roger Rowlett 71 Validating Structures Before a structure is deposited with the Protein Data Bank it is necessary to evaluate the proposed structure for its quality including consistency with typical known bond lengths and angles steric hindrance and appropriate hydrogen bonding networks Structures pdb files can be uploaded and evaluated at the www biotech ebi ac uk 8400 server It is recommended that you perform a complete check The following results should be especially scrutinized Typically you should be most concerned with any check results labeled bad e BPOCHK and BH2CHK these examine polar residues for missing hydrogen bonds Normally all polar residues i
65. gue Toshi Ohsumi a con conspirator in developing an open source parallelizable genetic algorithm for molecular replacement gets the nod for convincing me to try Phaser Some cover art was added to make the manual look less cheesy and more recognizable in the lab Abbreviations Used Some of the more common acronyms and abbreviations used in X ray crystallography used in this manual are listed here e SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis e PEG polyethylene glycol typically followed by an average polymer molecular weight e g PEG 400 has an average molecular weight near 400 e MPD 2 Methyl 2 4 pentanediol X Ray Crystallography Methods Roger Rowlett 3 Crystallization of Proteins Preparation Protein Samples Purification Protein samples should be as pure as possible for successful crystallization Protein that is gt 90 pure should be sufficient for commencing crystallization screens The more homogeneous the protein the more likely crystallization is to be successful Purity can be evaluated by SDS PAGE isoelectric focusing and or mass spectroscopy Sample storage Proteins are typically stored at 4 C or frozen at 80 C in a solution appropriate to maintain stability and activity Proteins solutions should be as concentrated as practical to enhance stability A stock protein concentration of 10 20 mg mL is typical for crystallization screening If protein is stored frozen it should be
66. h a final R free 0 20 or so depending on the quality of the original data The CNS components used in the refinement cycle are described below Simulating annealing is typically only carried out once Minimization and b factor refinement are carried out in the order listed after simulated annealing and after each model rebuilding cycle Simulated annealing This procedure shakes up the model to remove model bias and then does a whole molecule minimization to find an initial good fit to the experimental data Simulated annealing is carried out by the module anneal inp Required inputs are the pdb mt f and cv files the unit cell parameters and space group any extra t op or par files required the resolution range of the reflection data to be used typically all of the data and the output file typically anneal pdb Simulating annealing takes a very long time with large molecules and unit cells and is best run as an overnight job Minimization Minimization involves taking the input model and fitting it to the reflection data while conforming it to appropriate bond angles and distances for amino acid residues Minimization is carried out by the CNS module minimize inp Required inputs are the pdb mt f and cv files the unit cell parameters and space group any extra top or par files required the resolution range of the reflection data to be used typically all of the data and the output file typically minimize pdb
67. he command sym_sphere in the text or graphics window or choose the command gen_symmet ry on the user menu installed by 62 X Ray Crystallography Methods menu_rowlett odb The default radius for plotting symmetry related atoms is 10 A but this can be altered if desired Saving O sessions Saving your work in progress is not only desirable it s essential when working in O as it is known to crash unpredictably The entire state of your O project can be saved at any point by issuing the save_db command or by clicking on SAVE in the custom menu The first time you issue this command you will have to supply a filename O session files should be given the o extension to help identify them To retrieve and O session simply read in the appropriate o file at the initial prompt after starting O The entire state of O at the time of the save_db command including all odb files loaded at that time will be re instated SAVE your work frequently Basic Model Building Tasks in O There are several common tasks in rebuilding models to better fit the electron density maps Some of these are described here Typically after each refinement cycle the model is inspected for conformity to the electron density and modified as necessary to make it possible for the minimization algorithm to more easily find the best solution Mutating residues One of the first tasks to complete when a structure is being solved by molecular replacement is to change th
68. he same file that you are displaying in XDISP o box print defines the background area around each spot Set this value to about 3 times the radius of the spot size The command as written here is appropriate for images collected by an RAXIS IV system with 100 uM resolution image plates The format of this command may be slightly different depending on the detector system used 24 X Ray Crystallography Methods o spot radius enter the spot size in mm here The spot radius should be large enough completely enclose desired spots but not so large that spots overlap Start with a value of 0 7 mm o background radius defines a buffer zone between the spot and the background Typically set to 0 1 mm larger than the spot size If this line is omitted DENZO will assign the bare minimum buffer around the spot Before attempting to index the frame using DENZO identify major peaks in the image by pressing the Peak Sear button in XDISP Index the frame by entering the command denzo lt index gt index out and observe the XDISP window If things are working properly the peaks identified by the peak search should turn green and as refinement proceeds yellow Open a zoom window by pressing the Zoom Wind button in XDISP The position of the zoom window in the main frame can be changed by pointing with the mouse and clicking the middle button In the zoom window press the nt Box button so that you can observe the spot size and integrat
69. icity at high resolution where spot intensities are weaker Merging multiple data sets in CCP4i Frequently in protein X ray crystallography it is necessary to combine several datasets in order to solve a structure Such situations might include e combining several datasets at from different phi rotations of the same crystal This situation might arise from an interrupted data collection run where the initial data set was not sufficiently complete and for which it was impossible or impractical to resume the run exactly where it left off Combining two sets will allow the construction of a suitably complete data set e combining datasets from the same crystal using different camera distances This situation is very useful when a crystal has large unit cell dimensions and therefore closely spaced spots where it is difficult to collect a complete dataset which includes high resolution data as well as well resolved low resolution reflections In this case the low resolution data can be collected as a separate dataset with a longer camera distance allowing better separation of low resolution reflections Overlapping low resolution reflections are discarded in the high resolution data set e combining several datasets from different crystals of the same protein in the same space group This situation might arise when crystals have a limited lifetime in the X ray beam and no single data set is complete enough for structure solution Roger Rowlett 37
70. ile output by truncate to a format readable by EPMR The unix script mt z2epmr File 7 accomplishes this task The input hklin output hklout and log files should be edited as required This same task can also be accomplished in the CCP4i environment by choosing the task Convert from MTZ in the Reflection Data Utilities menu The CCP4i task window for carrying out the actions of File 7 are shown in Figure 12 Roger Rowlett 45 w convert From MTZ Job title mzzepmm Convert MTZ to USER format MTZin y181f23 Jytetrz3 integrate_sca amz o HKLout y181f23 Jyt6tre3 ntegrate_sca aepmm _ Browse _Vew Ouput Fortran format MTZ cokunns itt Same order as output file Label column F_all processed as FP Etse O e_ta column tabet Figure 12 Convert from MTZ task window Required fields are highlighted in color Data fields in MTZ file that are to be converted to user defined format are listed in the MTZ File Labels section File 7 mtz2epmr mtz2various hklin hical5b mtz hklout hical5 epmr gt mtz2epmr log lt lt eof various LABIN F FP OUTPUT USER 3I14 F7 1 END eof various EPMR also requires an additional file that contains information about the unit cell dimensions and the space group number This file should contain a single line in the format in which the values of a b c a B y and the International Tables space group number are entered separated by spaces The unit cell parameters
71. ill probably find the web interface of CNS the simplest to use when setting up CNS script files and CCP4i easiest for running CCP4 tasks More experienced users are more likely to modify previously written script files to run CNS or CCP4 tasks While both approaches are discussed here the CCP4 Coot packages are somewhat easier to manage and are better integrated and are recommended Preparing the first map is an exciting and expectant time You are either rewarded with immense joy of actually seeing clear electron density delineating the path of the main chain and positions of many side chains or you suffer the crushing disappointment of hash Either way here is how you generate the first map Initial model refinement using CNS Running CNS scripts The general method of running scripts is to type the command cns_solve lt filename inp gt filename log at the prompt This will run the script filename inp and output a log file to filename log You should always examine the log file to ensure that the program completed successfully Before you run CNS the first time it is usually necessary to enable the CNS modules by running the appropriate source command which is normally aliased to the command cnssetup Roger Rowlett 51 Constructing a cross validated reflection file This is an important step in your structure determination You are going to set aside a portion of your reflection data as a test data set that will not be used in the construc
72. in the asymmetric unit e To enhance the probability of finding an appropriate solution it is recommended that you check the box Final selection for rotation search peaks and enter 65 for percentage of top peak This will retain more possible solutions from cycle to cycle The default is 75 e It is also suggested that you increase the Packing tolerance by checking the appropriate box and allowing for 10 20 clashes This will slow the solution somewhat but will prevent the elimination of solutions that have molecular clashes between mobile and disordered portions of the search models when packed in the unit cell e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status Phaser jobs can take 2 24 hours depending on the complexity of the problem and the various selection criteria CCP4i jobs will continue to run even if you exit CCP4i and logout of your account e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly Preparing the first electron density map Preparing the first map and doing the subsequent refinements heavily uses CNS and the molecular display program O described later Alternatively you can use the CCP4 program Refmac for refinement and the molecular display program Coot Beginners w
73. ion boxes Examine all around the diffraction pattern using the zoom window In particular o Verify that the predicted spots match the observed spots If the preds are badly mismatched the most likely culprits are incorrect x beam y beam values incorrect distance or incorrect space group If the space group is suspect re index using space group P1 o Ifthe spots index well but you have more preds than spots then the mosacity is too high Lower the mosacity and re index If you have more spots than preds then the mosaicity is probably too low Increase mosacity and re index o Verify that the spot size is sufficient to enclose desired spots If spot size is too large many reflections will be rejected due to overlap with other spots If spot size is too small reflections will be rejected for spilling over into the background Examine the log file index out to determine the quality of the fit and verify space group assignment In particular o Examine the X value for the x direction y direction and partials y values near 1 indicate an acceptable fit y values over 4 should be cause for concern o Examine the space group fitting statistics The correct Bravais lattice space group is most likely to be the highest symmetry space group with a distortion index lt 0 5 Do not delete the index out file You will need the last 20 lines of this file to properly orient DENZO for the fitting of your entire data set as described below Rog
74. ions to output as PDB and MTZ files f NB MTZ files not output for all modes Component 1 protein molecular weight 2000 Number in asymmetric unit 3 Ensemble I Ensemble Mame hica15 Define ensemble via pdb file s PDB 1 Full path fprojectszyi 8101 epmr hica15 dfdimer pdb _ Browse ew Similarity of PDB 1 to the target structure sequence identity go o Allow search with altemative ensembles models for a single component of the ASU off Perform search using hica15 Number of copies to search for a Ehitist Ai another search M Final selection criterion for ROTATION search peaks percentage of top peak jeso _ Final selection criterion for TRANSLATION search peaks percentage of top peak ESI Packing tolerance number of allowed clashes Figure 14 Phaser task window set up for molecular replacement solution Required fields are highlighted in color e Inthe Define ensembles section provide an ensemble name and enter the filename of the search model as well as an estimate of the homology of the search model to the protein of interest Do not enter 100 if the two protein are not perfectly identical Underestimates are better than overestimates of homology 50 X Ray Crystallography Methods When using a wild type protein as a search model for site directed mutants a value of 90 is OK e Under Search Details enter the ensemble name and the number of copies to be placed
75. itable for safely freezing crystals Typically some quantity of cryoprotectant is added to a solution of artificial mother liquor or a solution of artificial mother liquor containing the appropriate amount of cryoprotectant is made up from scratch Some typical cryoprotectants and concentrations required to assure proper freezing protection in the worst case scenarios is given in Table 1 below In many cases a lower concentration of cryoprotectant that that listed in Table 1 is sufficient For example crystallization solutions already containing high concentrations of PEG may require little or no additional cryoprotection The minimum amount of cryoprotectant required can be determined by pipetting 10 uL drops of solution into liquid nitrogen If drops reliably freeze clear then the solution has sufficient cryoprotection for freezing protein crystals The choice of cryoprotectant will depend upon the crystallization solution composition If protein crystallization conditions already contain a cryoprotectant it is often ideal to simply increase the concentration to the appropriate value This is especially convenient for PEG containing solutions However PEGs have limited solubility in solutions that contain high concentrations of salt in this case one of the other cryoprotectants in Table 1 is more likely to be suitable Glycerol glucose or sucrose are very gentle to most proteins have high solubility in a large variety of solution and are often
76. l data on heavy atom derivatives of the same protein multiple isomorphous replacement by examining anomalous scattering of endogenous heavy atoms in the protein useful for certain metalloenzymes or selenomethionine substituted proteins or by using a starting model derived from a homologous protein molecular replacement This edition of the handbook will only treat the latter case molecular replacement There are many software suites available for the analysis modeling and refinement of protein X ray diffraction data The methods described here utilize the CNS suite Crystallography and NMR System and or the CCP4 Collaborative Crystallography Project suite and some other ancillary programs such as EPMR and Phaser Model building and examination of electron density maps is done using Alwyn Jones program O or Paul Emsley s Coot Preparing SCALEPACK data for analysis Converting scalepack data to CNS format Prior to refinement in CNS it is helpful to do a series of file conversions The first of these is to convert the SCALEPACK reflection file into the mtz format that CCP4 recognizes If you processed your data in MOSFLM and SCALA this step is not necessary The Linux script sca2mtz File 4 will do this To run this script the CCP4 suite must be enabled by running the appropriate source command which is typically aliased to the command ccp4setup The input file hklin the output file hklout the space group symmetry and the
77. lization conditions If more than half the drops are clear you should consider increasing the protein concentration and re screening If most of the drops have copious precipitate you should consider lowering the protein concentration and rescreening To save protein in the initial phase of screening it may be advisable to run only half of a screen at a time in a 24 well plate until you establish the appropriate protein concentration for efficient screening Conditions from the initial screen that show the most promise for crystallization should be further optimized in order to improve crystal form and size To find the best crystallization conditions pH precipitant concentration and protein concentration should be systematically varied This will require stock solutions of concentrates so that a variety of custom solutions can be constructed for optimization For example typical buffer stock solutions are 1M and pre adjusted to the desired pH Salt solutions are generally prepared to near saturation 1 4 M depending on the salt X Ray Crystallography Methods Crystal form can usually be further improved by exploring additives Preformulated additive screens can be purchased Hampton Research or selected additives stocks at 10x final desired concentration can be prepared and added at 10 volume to hanging drops Additional resources Bergfors Terese M 1999 Protein Crystallization Techniques Strategies and Tips International Unive
78. ll allow the adjustment of all the side chain torsion angles as well as the phi and psi angles of the main chain Before using the lego_side_chain command it is necessary to issue the lego_setup command first to initialize O for this purpose To choose from among common conformers of a side chain issue the command lego_side_chain or choose this command from the user menu and click on the residue you wish to alter Click in the fake dial box to update its menu and scroll through the possible rotamers by holding down the left mouse button while scrolling over the dial box Rotamer entry When you are satisfied with the selected Roger Rowlett 63 rotamer click on Yes in the user menu If you want to start over and discard changes click on No in the user menu For finer control over side chain conformation issue the command tor_residue or choose this command from the user menu and click on the residue you wish to alter The various adjustable torsion angles will appear on the selected side chain along with their current values The values can be very useful when using this command to manually flip Asn Gln and His side chains by 180 to improve hydrogen bonding contacts Click in the fake dial box to update its menu and several new items will appear corresponding to the various torsion angles in the residue The various torsion angles can be changed by holding down the left mouse button while scrolling over the appropriate dial box entry When y
79. lution data it may be advantageous to fix the solvent b value Typical solvent b values are between 100 400 A with 280 A being a commonly accepted optimal value e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verify that the job has run correctly Visualizing Molecules and Electron Density Maps in O O is a very powerful molecular visualization and model building program written by Alwyn Jones Uppsala University Sweden Using O it is possible to visualize the quality of fit of models to electron density data and also to interactively alter the model to better fit the data The latter of these activities termed rebuilding is essential to the refinement process Although refinement programs are very sophisticated it is not now possible for an automated refinement to find the best fit of model to data if the model is too far away from the correct solution The purpose of rebuilding is to position the model in a more appropriate starting point for refinement to do its magic Starting and customizing O The command for starting O is typically aliased to something easy to type For example ono9 might be the alias used to run O version 9 0 Usually the command is issued f
80. m Swing the goniometer to 90 and repeat Do the same for 180 and 270 orientations Repeat as necessary until the crystal is centered in the beam and rotates without wobbling in the center of the X ray beam You can check the accuracy of your alignment by looking for lateral displacement when the goniometer head is swung 180 i e you should check that the crystal is not laterally displaced when swung 0 180 C or 90 270 A properly mounted crystal is shown in Figure 5 When alignment is complete swing the goniometer to 0 and lock the goniometer head Remove all your tools and dewars from the hutch if you will need them during data collection otherwise they can stay inside Roger Rowlett 13 e Switch the X ray shutter from CLOSED to EXTERNAL exit the hutch and close the door e Start the R AXIS data collection software by entering the Unix command start amp at the prompt e Enter your data collection parameters For screening crystals it is useful to shoot three frames at 0 45 and 90 rotation about the axis to evaluate diffraction along different crystal axes If a practical camera distance is not known start with 200 mm This distance is sufficient to collect data to 2 5 A at the edge of the frame The oscillation range should be set to 0 2 2 0 or based on prior experience Exposure times per frame of 2 20 minutes are typical e Initiate data collection e Individual frames should be analyzed by DENZO MOSFLM or oth
81. mand is useful for monitoring the progress of programs that write log files while executing The tail f command must be terminated with ryc Special Linux Command Line Characters and Actions Linux has many special characters that make it easier to type commands Some of these are listed below with examples The tilde is used to designate your home directory For example if your home directory is home jdoe then the command 1s datafiles would list the contents of the home jdoe datafiles directory The dot is used to designate the current local directory For example the command cp home jdoe datafiles filename would copy the file filename from the home jdoe datafiles directory into your current directory using the same name The double dot is used to designate the directory one level up from your current directory For example the command cp datafiles filename would copy the file filename from the directory two levels up into your current directory The star is a wildcard character that can be used to select many similar files For example the command cp home jdoe datafiles osc would copy all files in the home jdoe datafiles directory ending with the characters osc into your current directory Be careful with wildcards especially when removing files You can always test your wildcard selection by doing an 1s command first If the 1s command lists the files you thought you selected you can change 1s
82. me jdoe directory into your local directory with the name my file Be careful with the mv command it will not check to see if you are copying over a current file with the same name The mv command is often used to rename a file in the local directory For example mv oldname newname would rename the file oldname to newname e kedit filename KEdit is a very nice KDE text editor that can be used to edit files and scripts If a filename is supplied it will open that file An alternative editor if installed is editpad e ps identifies the current processes running and their process identification numbers PID This command is most often used to obtain PIDs for the ki11 command e pwd print working directory This command returns the name of the directory you are currently located in e rm filename deletes a file from the local directory A useful but very dangerous variant of remove is rm r directory The rm r command is a recursive remove which 20 X Ray Crystallography Methods deletes a directory and absolutely everything that is in it including additional subdirectories and files within it Use with extreme caution rmdi r directory removes a subdirectory from within the local directory The subdirectory must be empty of files in order to remove it tail n filename displays the last n lines of the file filename A variant tail f filename will continuously follow the last lines of a file as it is being written This com
83. menu in the administration functions pane of the CCP4i window to verify that the job has run correctly Roger Rowlett 55 v Convert to MTZ amp Standardise Job title set_freer Import reflection file in MTZ format and create MTZ file W Create full unique set of reflections and generate FreeR data 181101 181101 sf mtz _ Browse vew yte fytem sfmaemz pamaen w sta jy1 81701 belonging to Project yi 81f01 Dataset name fan Set FreeR for fraction of reflections 0 05 _ Include systematic absences _j Extend resolution to 2 500 Figure 15 Task window for setting the FreeR flag in CCP4 reflection data Performing a REFMAC based refinement Refmac is a powerful and simple to use refinement package It will perform coordinate and b factor refinement of the model against the structure factor data and automatically write out phase and intensity information that can be used to construct electron density maps in Coot Refmac refinement is easily configured in CCP4i e Inthe CCP4i main task window select Refinement from the task menu and click on Run Refmac5 A task window will open Figure 16 e Enter a job name e g refmac and the input file name the sorted structure factor MTZ file for the entire dataset or the MTZ file from the previous refinement cycle Select restrained refinement with no prior phase information Filenames will be provided for the output data set and MTZ file or you can change th
84. mple cp home jdoe yourfile myfile would copy the file yourfile from the nome jdoe directory into your local directory with the name my file Be careful with the cp command it will not check to see if you are copying over a current file with the same name e df treport free block of space on disk drive To force display of free space in intelligible units kB and MB use df h e kill PID halts a process with the indicated PID process identification number This command is used to halt a program running in the background Sometimes kill is not adequate and a more severe variant kill 9 PID must be used The kill 9 command should normally be used only as a last resort e 1s tist directory This command will list the contents of the current directory You may add switches for additional functionality For example 1s 1 will make a long listing of files with additional file details including size and permissions On most Linux distributions 11 will carry out the 1s 1 command e mkdir directory creates a new subdirectory within the local directory e less filename displays the contents of a file a page at a time Tapping 1 or fF scrolls one page forward one page backward jumps to the beginning of the file EE jumps to the end of the file Type fe to quit e mv filename filename2 moves a file from one location to another For example mv home jdoe yourfile myfile would move the file yourfile from the no
85. mportant step in your structure determination You are going to set aside a portion of your reflection data as a test data set that will not be used in the construction of the model but will be used to independently measure how well your model fits the raw data The nature of the iterative procedure of modeling and refining biases the electron density map to conform to the model no matter how wrong it may be The set aside test data is your guard against falling too deeply into this trap Constructing a cross validation reflection files can be easily accomplished in the CCP4i environment e Inthe CCP4i main task window select Reflection Data Utilities from the task menu and click on Convert to MTZ and Standardize A task window will open Figure 15 e Enter a job name e g set freer and the input file name the sorted structure factor MTZ file for the entire dataset e A filename will be provided for the output data set or you can change it to something else e The crystal project and dataset names should be automatically recognized from your input file e Select a percentage of the data to set aside for the test Free R set The default value of 5 0 05 should normally be adequate e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status e When the job is finished examine the log file from the View Files from Jobs
86. n proteins are fully engaged in hydrogen bonding to something For each residue indicated to have missing hydrogen bonds examine the structure carefully Residues on the surface of the protein can usually be ignored as they are likely hydrogen bonded to water that is not visible in the electron density maps Modify the structure as required to correct missing hydrogen bonds if possible e ANGCHK any residues scoring gt 0 5 should be investigated as high scores indicate residues found in unusual conformers If the electron density clearly justifies the observed conformation no changes are necessary Otherwise modify the structure as required to bring the residue into conformity e HNQCHK this utility checks for Asn Gln or His residues that would establish better hydrogen bonding interactions if flipped 180 Use tor_residue in O to flip the required side chains 180 e Ramachandran plot examine the Ramachandran plot to determine if most residues except for mostly Gly are in the preferred b and y angle regions Typically at least 90 of the residues should be in the preferred regions Investigate any residues other than Gly that are in non preferred conformations and make corrections if necessary e WHATIF investigate all bad results in detail In particular examine bumps steric crowding to see if they are real or simply the result of large b factors Once structural problems have been resolved re refine the structure
87. ogether to perform reflection integration The following procedure is typical for integrating data e Copy your image file s typically osc into a local working directory e Create a subdirectory x to receive the integrated reflection intensities e Display the first frame by issuing the command xdisp raxis4 100 filename osc amp where filename osc is the name of the first image file An image of the diffraction pattern should appear e Obtain and edit the file integrate file 2 which contains instructions for DENZO integration This file should reside in the same directory as your images You should o Copy the last 20 lines of the previously saved index out file into the appropriate part of the integrate file These lines contain information about the crystal orientation and refined instrumental parameters in the first frame Delete any lines from this section that duplicate other instructions in the integrate file e g mosaicity settings o Enter the directory and filename for the reflection files inthe film output line o Choose the frames you would like to integrate by editing the sector instruction o Other settings should be edited to match those used to index the first frame e Before attempting to integrate using DENZO identify major peaks in the first frame by pressing the Peak Sear button in XDISP e Integrate the data by entering the command denzo lt integrate gt integrate out amp and observe the XDISP window If thing
88. on from the solvent region For the solvent mask calculation Increase VDW radius of non ion atoms by ra Increase ionic radius of potential ion atoms by 0 8 Shrink the area of the mask by oe after calculation For low resolution structures fix the B values of Babinet s bulk solventto Figure 16 Task window for running Refmac Roger Rowlett 57 e Important You should select the weighting term for the X ray structure factors carefully The default value of 0 3 is generally too high for typical data of 2 0 2 5 A resolution and will result in excessive distortion or mangling of the model To increase the weight of geometric restraints the X ray weighting factor should be decreased Typical values of the weighting factor are 0 05 0 20 depending on the resolution and quality of the data You should adjust the weighting factor until the RMSD of bond lengths and bond angles are 0 010 0 020 A and 1 5 2 0 respectively You can determine the RMSD of bond lengths and angles by examining the REFMAC log file This degree of geometric constraint should generate acceptable structures that are appropriately dependent on the observed X ray structure factors Once you have determined an adequate X ray weighting factor you may use that value for the remainder of the refinement e Select Babinet scaling This is typically more accurate than the default simple scaling and results in more reasonable b values for the protein structure For low reso
89. onvert to SFs 8 Wiison Piot Estimated number of residues in the asymmetric unita Use dataset name jas identifier to append to column labels W Include the intensities in the output MTZ file s Data Harvesting Create harvest file in project harvesting directory par Define Ouiput Datasets The input file contains a single dataset which will be transferred to the output file Crystal d44n01 belonging to Project d44n01 Dataset name nighres Scaling Protocol a Scale on rotation axis with secondary beam correction with isotropic Bfactor scaling Define scale ranges along rotation axis by rotation interval Secondary beam correction maximum number of spherical hannonics le independent Bfactors defined by rotation interval _ zo lt a Walk Ria BS ncwk on T RA KU 468 funntinn im anal NN ninun Ana Run Save or Restore _ dose Figure 9 The Scale and Merge Intensities task window in CCP4i Mandatory fields are highlighted in color e The scaling job is started by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status 36 X Ray Crystallography Methods e When the job is finished examine the scaling statistics by selecting View Files from Job View Log File from the administration pane In the log file window select Show Summary o The overall Rinerge Should be ve
90. ou are satisfied with the selected changes click on Yes in the user menu If you want to start over and discard changes click on No in the user menu Adjusting main chain conformation It will often be necessary to re orient the main chain as well as side chaings to better fit the electron density map This is most conveniently done using the grab_atom grab_fragment and grab_residue commands The grab_atom command will move individual atoms grab_fragment will move sidechains or mainchain atoms as a group and grab_residue will move the entire residue Similar manipulations can be carried out with the move_atom move_fragment and move_residue commands the main difference being that the move commands require the use of the dial box while the grab commands allow the use of the mouse to drag atoms fragments or residues about the screen The grab commands will be described here To grab an item to move about the screen issue the appropriate grab command or select it on the user menu then click on the item to be moved while holding down the mouse button The item can be moved in the x and y directions by simply moving the mouse Note these movements are in 2 dimensional screen coordinates only so it is usually wise to inspect the effects of your movement from several viewpoints to ensure that you have correctly placed the moved item in 3 dimensional space You may continue to click and drag the item as many times as you wish In addition you may
91. our image file An image of the diffraction pattern should appear If it looks usable proceed on to the next step e Obtain and edit the file index file 1 which contains instructions for DENZO indexing This file should reside in the same directory as your image files You should pay special attention to the following settings in the file and change them as necessary o wavelength if not using a rotating copper anode source 1 5418 A change to the appropriate value o x beam and y beam obtain the correct values for the X ray beam center from the latest log book entry for the instrument It is difficult to properly index reflections unless these values are accurately known o distance enter the camera distance here This value must be known accurately in order to properly index reflections o mosaicity a measure of the disorder of the crystalline lattice Start with a value of 0 4 0 7 degrees o raw data file enter the image file name here with the symbol used to indicate the number and position of the numerical part of the filename that tracks the frame number o space group if known enter the space group here if unknown start with the lowest symmetry space group P1 o oscillation range enter the oscillation angle used here typically 0 2 2 0 This value must be known accurately in order to properly index reflections o sector enter the frame number of the file you would like to index Normally it should be t
92. quid nitrogen until it stops bubbling vigorously Use the crystal wand to unscrew the crystal cap with the mounted crystal Keep the crystal cap under the surface of the liquid nitrogen at all times Remove the empty vial and vial clamp from the liquid nitrogen and set aside Keep the crystal cap totally submerged in liquid nitrogern during this and the next two steps Insert the cryo tongs into the liquid nitrogen and hold them there until all bubbling stops This may take 30 60 seconds The tongs will cool faster if they are held open during cooling It is important that the cryo tongs are fully cooled before proceeding While keeping both tongs and crystal cap under the surface of the liquid nitrogen open the tongs grasp the crystal cap and remove it from the crystal wand The wand can now be removed from the liquid nitrogen and set aside Quickly remove the cryo tongs and the attached crystal cap from the liquid nitrogen and place the cap on the magnetic head of the goniometer The tongs should be oriented so that when they are opened to release the cap the crysostream can blow into the opening between the two halves of the tongs This will prevent ice ring formation or crystal melting Immediately adjust the goniometer head so that the crystal is centered vertically in the beam and properly centered in the cryostream Unlock the axis of the goniometer and swing it to 0 Adjust the goniometer with the hex key until it is centered in the bea
93. r images derived from the Oxford Diffraction Excalibur system o backstop radius 4 0 o gain 1 2 o end ba MOSFLM version 6 2 4 Image Display HlEdits allowed Main menu s x EJ Hrca_n44N01_3t 1_1_001 img Min 1 Max 5367 axl 48 ax2 515 Overlay 7 on Contrast m Colour 7 Black on white Mag 7 x4 Read image Find spots Edit spots Clear spots Select images Autoindex Estimate mosaicity Predict Clear prediction Adjust Refine cell Integrate Strategy Keyword input Find hkl Pick Measure cell _OO0000000 Circles Beam backstop Save Exit WONODOONNUN oo Output Pixel X Y 1025 0 XC YC mm 125 4 0 0 Resolution 0 00 a Indices 0 Aft int ti No a RE i 0 00 width 0 00 Timeout mode 0 00 0 00 0 00 Figure 7 MOSFLM graphical window Processing parameters are displayed on the left main menu is in the middle image pane is on the right e Commence autoindexing by clicking on Autoindex in the main menu You will be prompted with a series of questions shown below with typical answers Comments are given in italics 32 X Ray Crystallography Methods Do you wish to continue Y the default can always be accepted by typing pfa Do you want to find spots manually N Do you want to add spots manually N Do you want to try the new autoindexing Y Do you want to fix detector distance Y Do you
94. ranged so that the output filename for one module is the input filename for the next module That is geneasy inp uses input pdb as an input file and outputs geneasy pdb and geneasy mtf minimize inp uses geneasy pdb and geneasy mtf as input files and outputs minimize pdb bgroup inp uses minimize pdb and geneasy mtf as inputs and outputs bgroup pdb etc 54 X Ray Crystallography Methods File 10 CNS refine script Roger Rowlett Feb 2003 This script takes a PDB file and carries out CNS Minimize Bgroup and Bindividual refinement and generates an output PDB file and both 2FoFc and FoFc maps based on the refined model kkk K KNOTE NOTE NOTE NOTE NOTE Edit input PDB filename in second line Edit output PDB filename in last line Run cnssetup prior to execution Run ccp4setup prior to execution kkk K KNOTE NOTE NOTE NOTE NOTE rm geneasy pdb minimize pdb bgroup pdb bindividual pdb map cp hica01 3c pdb input pdb cns_solve lt geneasy inp tee geneasy log cns_solve lt minimize inp tee minimize log cns_solve lt bgroup inp tee bgroup log cns_solve lt bindividual inp tee bindividual log cns_solve lt 2fofc inp tee 2fofc log cns_solve lt fofc inp tee fofc log map_to_omap 2fofc map map_to_omap fofc map cp bindividual pdb hica01 4 pdb Se SR OSE lt gt lt gt Refining Structures using CCP4 Constructing a cross validated reflection file This is an i
95. re dataset Roger Rowlett 43 e A list of available fields will appear in the MTZ File Labels section The only fields typically required for structure solution are FP and Sigma which correspond to the structure factor and its standard error Set all other fields to Unassigned using the drop down menus e Select an output file name It is suggested that you make the file extension fobs to be consistent with the instructions in this methods manual e Start the job by selecting Run Run Now at the lower left of the task window The job will be entered into the job list in the CCP4i window and you can monitor its status Convert From MTZ Job title Convert MTZ to CNS format MTzin d44n01 jd4ano1 all sf mtz Browse View HKLout d44n01 d44no1 al sfhkl Browse View F_all Sigma SIGF_all F all SigmaPH SIGF_all F all FOM Unassigned L L DANO all SIGDP SIGDANO all F F_all _ SIGF SIGF_all F F_all SIGF SIGF_all PHIC Unassigned Weight Unassigned Best PHI Unassigned FreeR Unassigned m L L L L Figure 11 Convert from MTZ task window Required fields are highlighted in color Available data fields in MTZ file are listed in the MTZ File Labels section e When the job is finished examine the log file from the View Files from Jobs menu in the administration functions pane of the CCP4i window to verif
96. re described here Typically after each refinement cycle the model is inspected for conformity to the electron density and modified as necessary to make it possible for the refinement program to more easily find the best solution Mutating residues One of the first tasks to complete when a structure is being solved by molecular replacement is to change the mismatched residues in the search model to conform with that of the target molecule In Coot choose Calculate Mutate Residue Range In the dialog box choose the protein chain and the residue number or range to be mutated and type in the one letter amino acid code s for the mutation If desired you can autofit the mutated residue to the electron density map upon mutation by checking the appropriate box Ajdusting side chain conformation Open the refinement task menu by selecting Calculate Model Fit Refine You now have several options to adjust side chain conformation on the task menu Auto Fit Rotamer will select the best fitting side chain rotamer from a library of commonly observed conformations This may be a good first attempt in some situations You may also elect to interactively select a rotamer from the library by selecting Rotamers from the task menu To further refine this solution automatically you can select Real Space Refine Zone and then click twice on any atom in the side chain to define the side chain as the refinement zone A dialog box will offer you the choice to ac
97. riate file name Both 2F F blue and F F positive green and negative red electron density maps will be automatically displayed The graphical viewing environment of Coot is shown in Figure 18 By default the 2F F map is contoured at approximately 1 56 and the F F is countoured at approximately 3 00 The countour settings can be changed by rolling the center wheel of the mouse To select which map will be re contoured by default select HID ScrollWheel Attach ScrollWheel to which map Navigating and inspecting a molecule in Coot The molecule can be manipulated on the screen with the mouse Press the left mouse button and drag to spin the molecule To zoom press the right mouse button and drag To slab cut away the molecule press r and the right mouse button and drag up and down To navigate to a particular atom select Draw Go To Atom to open up the navigation window and select the desired protein chain residue and or atom desired To recenter on an atom visible on the screen middle click on it To navigate to the next residue in the sequence press the space bar Generating symmetry atoms and non crystallographic symmetry traces It is frequently useful to generate symmetry related atoms in the displayed model in order to observe interactions at protein protein interfaces or to get a more accurate view of an interfacial active site etc To display symmetry atoms choose Draw Cell amp Symmetry and tick Yes in th
98. right carat except that it will append rather that overwrite data to an output file For example the command myprog lt input txt gt gt output txt amp would launch the program myprog in the background accept input from the text file input txt rather than the default keyboard and append results to the file output t xt rather than the default screen If output txt does not already exist it will be created The pipe is used to feed the output of one program into another For example the command myprog lt input txt tee output txt would launch the program myprog accept input from the file input t xt and send the results to the program tee which sends output to both the screen and the file output t xt This example is another way to monitor the progress of an executing program while saving the output to a text file 22 X Ray Crystallography Methods Customizing Your Linux Environment It is possible to customize your Linux environment to make it easier to navigate through your directories and projects To customize your environment edit the tcshrc file in your home directory Commands in this directory will be executed each time you open a new shell window The following types of commands are useful to have in your tcshrc file e set history 100 this setting allows the last 100 commands to be remembered You can call them up at the prompt by pressing the f key as described previously e alias name command
99. rm reflection indexing The following procedure is typical for indexing a single frame of data Copy your image files s typically img into a local working directory Navigate to this directory and configure CCP4 by issuing the command ccp4setup then start MOSFLM by issuing the command ipmos f lm The MOSFLM prompt should appear Load an image into MOSFLM using the image and go commands For example to load the image file xyz 001 img issue the commands image xyz 001 img then go The MOSFLM graphical window should appear with your loaded image file Figure 7 For most image file formats MOSFLM can read camera distance and X ray wavelength data directly from the file Check the Processing Parameters pane to verify these values are correct Processing parameters can be changed by clicking on the item you want to change and typing the desired value Enter accurate values for Beam X and Beam Y in the Processing Pane by clicking on these items and typing the desired value Before indexing it is helpful to set the detector gain and backstop radius This is conveniently done by selecting Keyword input on the main menu Keyword input is terminated by typing end after the last keyword entry The backstop radius should be just large enough to exclude the central region of the image blocked by the beam stop the detector gain should be set to a value suggested by the manufacturer The following Roger Rowlett 31 keyword commands are typical fo
100. rom the local directory from which you are working so that you do not have to specify complete paths to your data files O will ask for the locations of several files on startup and normally the defaults should be accepted If you want to read in a previously saved O session provide the file name of a session file at the prompt to the first question Note if running O in Windows you should always read in the file odat odb at the first prompt to tell O where to find its internal data files The file odat odb is described later O is rarely run without a great deal of personal customization which is done by including a series of series of custom files in your working directory Typical files include a personal 58 X Ray Crystallography Methods menu special scripts to automate commonly used tasks O database files to modify how O works and a list of commands to execute on startup The last of these must be put into a file named on_startup It is a good idea to give all O database files the extension odb so that you will know their intended use File 11 menu_rowlett odb MENU 41 24 lour_text red STOP lour_text white Save_DB colour_text magenta Clear_flags colour_text green Yes colour_text red No colour_text cyan Centre_ID Clear_ID colour_text yellow Dial_previous Dial_next colour_text cyan Lego_CA Lego_side_ch Water_add colour_text yellow Grab_atom Grab_fragment Grab_residue Move_zone colour_text
101. rsity Line La Jolla CA Hampton Research http www hamptonresearch com A practical guide to protein crystallization Mark Knapp http www structure llnl gov crystal lab crystall htm The Protein Crystallization Page Terese Bergfors http xray bmc uu se terese X tal Protocols Johan Zeelan http www mpibp frankfurt mpg de johan zeelen xtal html How to grow protein crystals http www ccp14 ac uk ccp web mirrors IInlrupp crystal_lab cystalmake html Protein crystallography course http www structmed cimr cam ac uk Course Crystals intro html Roger Rowlett 7 Soaking Mounting and Freezing Protein Crystals Most X ray crystallographic data collection is done at low temperature typically 100 K to minimize degradation of the crystal by free radicals generated by the X ray beam This is especially important when using intense synchrotron X ray sources In order to prevent crystals from cracking when frozen it is necessary to treat protein crystals with a cryoprotectant prior to freezing In the presence of a cryoprotectant the protein and its thin layer of surrounding mother liquor will form an amorphous glass in which the crystal suffers minimal damage and retains maximum X ray diffraction properties Screening for a suitable cryoprotectant Unless the optimum crystallization conditions already contain a sufficient quantity of cryoprotectant it will be necessary to experimentally determine solution conditions su
102. ry low typically 0 05 for an excellent data set Overall Rinerge Values gt 0 10 are cause for concern For a typical data set the Rinerge values by shell should increase monotonically from low to high resolution shells You may consider disregarding high resolution data with Rinerge gt 0 40 and re scaling with reduced resolution limits o The overall I o 1 value should typically be 20 An overall I o T lt 10 is cause for concern For a typical data set I o I should decrease monotonically from low to high resolution shells You should disregard shells with I o 1 lt 2 and re scale with reduced resolution limits o Examine the completeness of the data set Data that is 85 90 complete should be sufficient to solve a structure although more completeness is better if practical A quality data set will also have approximately the same degree of completeness in each shell with perhaps a monotonic fall off at high resolution where spot intensities are weaker or are limited to the corners of the images Gaps in completeness in low or mid resolution shells may indicate problems with ice rings and or integration o Examine the multiplicity of the data set A typical data set will have an average multiplicity of 4 This is a measure of the average number of times a reflection intensity Z or its Friedel mate Znz has been independently measured Higher multiplicities will result in a more precise data set There may be a fall off in multipl
103. s lower than in the well solution water evaporates from the drop increasing the concentration of both protein and precipitant until the drop is in equilibrium with the well solution The concentration of protein and precipitant in the drop occurs slowly and gradually favoring crystallization over precipitation drop Figure 1 Hanging drop vapor diffusion X Ray Crystallography Methods Preparing crystallization trays Crystallization trials are conveniently performed in 24 well pre greased crystallization trays fig 2 Prior to setting trays carefully organize your solutions and record in your notebook the crystallization conditions to be used in each well The following protocol is typical Obtain a pre greased 24 well crystallization tray and a box of 22 mm siliconized cover slips If setting trays at 4 C allow the tray and all solutions to equilibrate before proceeding Fill the wells of the tray with the appropriate precipitant solutions Remove a coverslip from the box taking care to handle only by the edges Pipet 1 uL of protein on to the cover slip taking care not to introduce bubbles Pipet an equal volume of precipitant solution from the corresponding well into the protein drop and gently mix by pipetting up and down a few times Immediately place the cover slip over the well press down gently and twist 45 to ensure a good seal Repeat for remaining wells Immediately after preparing the plate plac
104. s are working properly the peaks identified by the peak search should turn yellow as they are identified overloads peak overlaps and spots too large to fit in the current spot size are flagged in red e Observe the output stream from DENZO in real time by typing the command tail f integrate out This will allow you to examine the output as integration proceeds e Open a zoom window by pressing the Zoom Wind button in XDISP The position of the zoom window in the main frame can be changed by pointing with the mouse and clicking the middle button Place this window on a convenient portion of the image so that you can follow the progress of the integration The command as written here is appropriate for images collected by an RAXIS IV system with 100 uM resolution image plates The format of this command may be slightly different depending on the detector system used Roger Rowlett 27 File 2 integrate title Refine All Images format raxis4 100 INSERT CRYSTAL ORIENTATION PARAMTERS HERE cassette rotx 0 01 roty 0 07 rotz 0 00 2 theta distance 179 33 x beam 150 473 y beam 151 274 y scale 1 00340 film rotation 180 000 skew 0 00025 crossfire y 0 001 x 0 051 xy 0 043 goniostat single axis goniostat orientation 0 000 0 000 motor axis 0 000000 1 000000 0 000000 profile fitting radius 25 00 resolution limits 30 0 2 00 wavelength 1 54180 monochromator 0 000 spindle axis 0 0 1 vertical axis 1 0
105. s not included with the CNS distribution can usually be downloaded from the HIC UP server Fine tuning the molecular replacement solution The molecular replacement solution should be fine tuned by adjusting the position of the model including the subunits independently of each other via a rigid body refinement This is accomplished by the CNS module rigid inp Required inputs for rigid inp are the pdb mtf and cv files the unit cell parameters and space group any extra top or par files required the resolution range of the reflection data to be used typically 15 0 4 0 A the segid names to be minimized typically all of them and the name of the output file typically rigid pdb The highest resolution shell should not be set too low a value else the refinement may not be able to move the model far enough to find the global mininum best fit Rigid body refinement needs only be run this one time It does not have to be run again during the refinement procedure Normally the R factor will decrease by 5 or more during rigid body refinement and this is usually a good sign that things are going well You should also monitor your R test vs your R free values at this point and after all subsequent refinement steps R test is the residual experimental data used for refinement not explained by your model R free is the residual test data the 10 you put aside in your cv file that is not explained by your data Normally R test is lower than R
106. sid odb the 2F F map and color codes both the positive and negative density in the F F map according to its deviation in o It also positions all the menus on the screen so as not to interfere with the visualization of the molecule of interest File 18 on_startup read menu_rowlett odb read resid odb win_open user_menu 0 9 1 0 win_open object_menu 1 20 0 7 win_open dial menu 1 20 0 2 fm_file 2fofc omap 2fofc C2 fm_file fofc omap fofc C2 fm_file fofc omap fofc C2 Fm_setup 2fofc 20 1 1 medium_blue Fm_setup fofc 20 3 5 white 4 cyan 3 blue Fm_setup fofc 20 3 3 red 4 orange 5 yellow window_open density_1 55 9 window_open density_2 0 05 9 window_open density_3 0 65 9 Loading a molecule into O A molecule can be loaded into O for inspection by issuing the command pdb_read Commands can be entered in the graphics window or in the text window Extended command sessions are best done in the text window Note if running the Windows version of O all commands must be entered into the text window When prompted supply the filename to read in and a molecule name 6 letters or less that you will use to identify the molecule in O To make the molecule visible type mol molname where molname is the name you supplied in pdb_read To render the entire molecule type zone end If the molecule does not appear it is probably not centered in your viewing area Center the molecule by using a command such as ce_atom
107. stal cap under the surface of the liquid nitrogen at all times e Plunge the vial clamp and attached vial into the liquid nitrogen and hold it there until it stops bubbling e While keeping both vial and crystal cap submerged in liquid nitrogen use the crystal want to screw the crystal cap on to the vial Set the crystal wand aside e Place the vial containing the crystal into the appropriate cryocane and return it to the tall dewar The cryocane should be returned to its storage dewar when convenient e Power down the cryostream controller and attach the house nitrogen line to the cryostream head This will prevent condensation and ice formation in the cryostream head e Remove your tools and supplies from the hutch 16 X Ray Crystallography Methods Roger Rowlett 17 Bare Bones Linux Because so many X ray crystallography data analysis and protein modeling and refinement programs are written for the Unix Linux platform some familiarity with the Linux operating system is necessary to do protein crystallography The following information is not intended to be an exhaustive Linux tutorial rather it is intended to be the bare minimum information required for starting ending and navigating a Linux session using a workstation in the Colgate University Department of Chemistry Protein X ray Crystallography Computing Facility The latest information concerning the status of the Computing facility is available at http departments colgate
108. talline state Normally one should download for each non protein molecule a clean pdb file the CNS topology and parameter files for guiding refinement in CNS the O connectivity entry and the O refi dictionary entry if you intend on using refi_zone on the hetero molecule The O connectivity entry should be added to a copy ofall dat and stored in your working directory When O prompts for the location of all dat during startup point to the modified copy The refi dictionary entry should be added to the bonds_angles O datablock and saved in your local directory according to the file instructions In all CNS refinement files it will be necessary to add references to the topology and parameter files for each hetero molecule Some ligands already handled by CNS such as monocations some monoanions phosphate and sulfate need not have separate topology and parameter file references in CNS scripts Adding ligands to a protein The simplest way to accomplish this is to use pdb_read to load the pdb file of the desired ligand into an existing O session with the protein and electron density map displayed Select and draw the molecule Use grab_residue to drag the ligand to the appropriate location and fit it into its electron density Once the ligand s is are in place write out a pdb file of the ligand s using the pdb_write command Use MOLEMAN2 to edit the ligand pdb file and give it a unique segment id Then the protein pdb file and the li
109. tion of the asymmetric unit u Component 1 protein molecular weight szoo0 Number in asymmetric unitfi Edit list _ Define another component 1 Additional parameters Figure 13 Phaser task window set up for Matthews probability estimation Required fields are highlighted in color Performing molecular replacement calculations in Phaser Phaser is a fast highly automated program for finding molecular replacement solutions for multiple protein molecules search models in an asymmetric unit Phaser is conveniently run in the CCP4i environment e Inthe CCP4i main task window select Molecular Replacement from the task menu and click on Phaser A task window will open Figure 14 e Enter ajob name e g phaser and the input file name the sorted structure factor MTZ file for the entire dataset e Under Mode for molecular replacement select automated search e Under Composition of the asymmetric unit choose protein and enter the molecular weight of the search model and the number of these molecules you expect in the asymmetric unit based on Matthews probability analysis Roger Rowlett 49 4Maximum Likelihood Molecular Replacement Job title phaser Mode for molecular replacement automated search MTZ in yam fysastmj ce a F F all SIGF SIGF all Resolution range 29 641 A to 2 500 A Space group read from mtz file C2 Run Phaser with the mtz space group Number of top solut
110. tion of the model but will be used to independently measure how well your model fits the raw data The nature of the iterative procedure of modeling and refining biases the electron density map to conform to the model no matter how wrong it may be The set aside test data is your guard against falling too deeply into this trap Modify the file make_cv inp so that it will set aside 5 10 of your reflections as test reflections If your data is nearly complete gt 95 you should use the maximum value 10 The input file should be the fobs file you created previously The output file should be given the cv extension to uniquely identify it This cv file will be used to do all subsequent refinements Generating CNS topology files CNS requires in addition to a PDB file a molecular topology mt file that contains information about molecular connectivity and geometrical constraints necessary to guide the refinement You must generate a new mt f file whenever you have add or delete atoms from your model It is typical to generate a new mt f file at the beginning of each refinement cycle To generate an mtf file edit and run the script generate_easy inp The required input is a pdb file and the outputs are a new pdb file and an associated mt file Be sure you have included the necessary topology top and parameter par files for ions water and hetero compounds included in your model Hetero compound pdb top and par file
111. to 260 fit crystal mosxx 1 to 280 Output add partials 1 to 280 output file hical5 sca output anomalous eof scalepack e Examine the scaling statistics paying particular attention to the following items o The overall Rzym should be very low typically 0 05 for an excellent data set Overall Rsym values gt 0 10 are cause for concern For a typical data set the Rsym values by shell should monotonically increase from low to high resolution shells You should probably disregard high resolution data with Rsym gt 0 30 and should re adjust resolution appropriately in the scale file o The overall I o 1 value should typically be x20 An overall I o I value lt 10 is cause for concern For a typical data set the I o I value should monotonically decrease from low to high resolution shells You should probably disregard shells with I o I values lt 2 and should re adjust resolution appropriately in the scale file o Examine the overall completeness Data that is 85 90 complete should be sufficient to solve a structure although more completeness is better if practical A quality data set will also have approximately the same level of completeness in each shell perhaps with a monotonic fall off at high resolution where spot 30 X Ray Crystallography Methods intensities get weaker Gaps in completeness in low or middle resolution shells may indicate problems with ice rings and or integration If you are scaling the final d
112. toms in the model to define a range of atoms to be regularize Select Yes on the user menu to accept the refinement or No to cancel Alternatively a command can be issued in the text or graphics window For example the command refi_zone hica a44 a46 would regularize all atoms in chain A of the molecule hica for residues 44 46 Again select Yes to accept and No to cancel the refinement Writing out a PDB file After modifying a model it is likely that you will want to write out a new PDB file reflecting the changes you have made in the model To write out a PDB file issue the command pdb_write and specify a file name when prompted Give the filename a pdb extension to help identify it for later use Adding ligands and cofactors to a model in O Many proteins contain non protein cofactors such as metal ions and coenzymes In addition crystallized proteins may tightly associate with buffer molecules inorganic ions and precipitant molecules It is often quite desirable to account for the electron density of these substances by adding them to the model Using HIC UP While it is possible to manually edit pdb files to do this normally it is much more convenient to download an existing geometry optimized model in pdb format A good source for such files is the HIC UP server found at http xray bmc uu se hicup This server contains over 4000 commonly encountered non protein molecules that have been found to associate with proteins in the crys
113. ue tolerable before excessive spot overlap occurs Exposure times should be long enough to accurately measure the intensities of the highest resolution spots commensurate with the length of data collection time you have e Initiate data collection e After a 100 or more frames have been collected they can be integrated and scaled by DENZO and SCALEPACK to assess the quality and completeness of data as described in the section on Diffraction Data Analysis When data collection is sufficiently complete you can stop data collection and shut down the instrument as described below Shutdown Procedure The X ray diffractometer should be shut down in an orderly fashion in order to maximize the life of the X ray source and prevent problems with the cryo system The following procedure should be employed e Obtain and don comfortable Thinsulate gloves to protect your hands from frostbite e Obtain two tall dewars and fill them with liquid nitrogen Place the appropriate cryocane s to store your retrieved crystal in one of the dewars e Obtain a vial clamp crystal wand and cryo tongs for handling your crystals and take all the abovementioned items to the X ray collection area e Ensure that data collection has stopped If necessary use stop data collection manually using the emergency stop button in the data collection software Do not enter the hutch until you have verified that the X ray beam shutter has been closed e Enter the hutch and imm
114. up Examine the log file to ensure that the program ran satisfactorily before proceeding File 5 trunc truncate hklin hical5a mtz hklout hical5b mtz gt trunc log lt lt eof truncate TRUNCATE YES NRESIDUES 1374 END eof truncate Converting MTZ files to CNS format The final step before beginning data analysis in CNS is to convert the reflection file data of a CNS compatible format This will not be necessary if using CCP4 to do structure refinement The Linux script mtz2fobs File 6 does this conversion The input file hklin the output file hklout and the log filename should be edited as required To run the script type trunc at the prompt To run this script the CCP4 suite must be enabled by running the appropriate source command which is typically aliased to the command ccp4setup Examine the log file to ensure that the program ran satisfactorily before proceeding The fobs file is the starting point for data analysis File 6 mtz2fobs mtz2various hklin hical5b mtz hklout hical5 fobs gt mtz2fobs log lt lt eof various OUTPUT XPLOR LABIN F FP SIGF SIGFP END eof various This task can also be carried out in the CCP4i interface by the following steps e Inthe CCP4i main task window select Reflection Data Utilities from the task menu and click on Convert from MTZ A task window will open Figure 11 e Enter a job name e g mtz2fobs and the input file name the sorted structure factor MTZ file for the enti
115. voltage to 50 kV and 2 increasing the current to 100 mA These steps should be carried out in this order and it is precautionary to increase both settings gradually as you bring up the source 12 X Ray Crystallography Methods Collecting diffraction screen data The following steps are typical for collecting crystal screens Obtain and don comfortable Thinsulate gloves to protect your hands from frostbite Obtain two tall dewars and fill them with liquid nitrogen Extract the cryocane s with the appropriate crystals and place in one of the dewars Obtain an empty crystal cap and loop the same size and type as those you will be using for data collection Obtain a vial clamp crystal wand and cryo tongs for handling your crystals and take all the abovementioned items to the X ray hutch Move the cryostream head back to allow sufficient clearance for mounting crystals on the goniometer without touching the X ray source beam stop or cryostream head Mount the empty crystal cap and loop on the goniometer head and adjust the height so that the loop is centered in the X ray beam and cryostream Failure to do this prior to mounting your crystals may result in their melting before they can be centered in the cryostream Remove the empty cap and loop when adjustments have been completed Using the vial clamp remove the desired vial from the cryo cane and keep it under the surface of the liquid nitrogen Immerse the crystal wand into the li
116. y has an important use in Linux as a paste command This is especially useful when editing command lines with long file names You can select text in virtually any Linux application including the terminal window by holding down the left mouse button and dragging To paste this text into the command line or another Linux application move the cursor to the insertion point and click the middle mouse button Input and Ouput Redirection Linux allows the user to redirect information from the keyboard or screen defaults for input and output to files or even other programs using redirection commands A listing of common redirection commands is given below with examples The left carat lt is used to redirect input For example the command myprog lt input txt would launch the program myprog and accept input from the text file input txt rather than the default keyboard Running programs using input scripts rather than the keyboard is a very common way of executing programs in Linux The right carat gt is used to redirect output For example the command myprog lt input txt gt output txt 6 would launch the program myprog in the background accept input from the text file input t xt rather than the default keyboard and output results to the file output txt rather than the default screen You could monitor the progress of myprog if desired by issuing the command tail f output txt The double right carat gt gt is like the
117. y that the job has run correctly e The resulting fobs file is now ready for use in structure solution Obtaining initial phases by molecular replacement The simplest method of obtaining phase estimates for X ray diffraction data analysis is molecular replacement which involves building a provisional model of the target protein based on the structure of a highly homologous protein and placing it in the appropriate orientation in the unit cell The initial phases are calculated based on the positions of all the atoms in the 44 X Ray Crystallography Methods molecular replacement model and such phases are often sufficient to obtain a usable electron density map that can be used to refine the structure of the target protein Two excellent tools for solving structures by molecular replacement are EPMR and Phaser both of which are detailed here Constructing a molecular replacement model For any molecular replacement solution it is necessary to construct a reasonable molecular replacement search model Select a molecular replacement protein that is as homologous as possible to the target protein and examine a sequence alignment of the two proteins A molecular solution replacement may be possible if the proteins are more than 30 identical The molecular replacement protein should be modified as follows to make it a similar as possible to the target protein e Ifthe molecular replacement protein has extra residues either internally or at

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