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1. To read through all log messages in the status bar simply use the arrows in the right corner 13 Computed 9 tunnel s from the origin with ID Computed0 in 0 6s Computed 9 tunnel s from the origin with ID Computed9 in 0 5s Figure 17 The Status bar of the MOLE 2 0 GUI provides a log of all calculations performed 4 4 Interpreting results In the case of tunnels and pores the GUI version of MOLE 2 0 also offers a useful way to immediately visualize and export the physicochemical properties of the identified voids These properties influence the size and type of ligands that can pass through a tunnel the direction of travel and how long it takes for the ligand to go through Such information becomes available via the Details button of each tunnel or pore from the Results panel For each tunnel you can open a window containing its physical properties Figure 18 The window consists of two tabs that you can switch between At the top of the window you can find the export property menu Various kinds of properties see below can be copied to clipboard in different formats and used for further analyses Such data can be exported also via the Export section in the Result panel for details please see chapter 4 5 1f4w 1TQN X Hide Controls Specific Point Residue s ae X x CSA Database 2 1TQN Tunnel 2 in cavity 1 Copy Profile Lining Props PDB XML J A o gt Start Points 15 Results 7 mb Y Cavitie
2. WebchemistyTunnels core dli 2013624PM Application erens 1598 E moleexe Datemodied 15 2013 1237 AM ate created 1 16 2013 1150 PM Application Size 654 MOLE 2 0 Sehnal D Berka K Banas P Navratilova V Pravda L lonescu C M Svobodova Varekova Koca J Olyepka M MOLE 2 0 Tool for Analysis of Biomolecular Channels in preparation MOLEONline 2 0 Rerka K Hanak O hnal D Ranas P Navrallova V Jaiswal D lonescu CM Svobodova Varekova R Ka a J Otvenki Figure 4 Start the MOLE 2 0 GUI by rumning the file mole exe PMI MOLE 2 13 18 AVID 4 Ak MOLE gt a structure from your hard drive a previously saved workspace 1TQN from the PDB Database Figure 5 Start screen of the MOLE 2 0 GUI A Functionality The MOLE 2 0 GUI based application allows for the rapid and automated calculation of cav ities tunnels and pores in biomacromolecules from the smallest polypeptides through glycans and lipids to the largest protein nucleic acid complexes found in the Protein Data Bank More over it comes with a built in molecular viewer allowing for immediate visualization of results as well as interactive refinement of the calculation A remarkable advantage of MOLE 2 0 is the fact that it allows you to interactively tweak the tunnel detection algorithm such that the results are immediately available for inspection and comparison Another useful feature of the GUI based version of MOLE 2 0 i
3. gt lt Boundary gt lt Residues gt 1 ASN A 2 LYS A 5 GLU A lt Residues gt lt Properties Charge 0 NumPositives 123 NumNegatives 123 Hydrophobicity 0 59 Hydropathy 1 29 Polarity 16 07 Mutability 80 gt lt Boundary gt lt Inner gt lt Residues gt 3 TYR A 4 PHE A lt Residues gt lt Properties Charge 35 NumPositives 71 NumNegatives 106 Hydrophobicity 0 28 Hydropathy 0 05 Polarity 6 81 Mutability 78 gt lt Inner gt lt Cavities gt Each empty space consists of a Boundary and an Inner part The boundary contains all the residues at the interface between the cavity and molecular surface The remaining residues belong to Inner part Note that the cavity type Void does not contain any residues in the Boundary part since the difference between void and cavity is that void does not have any connection with the molecular surface The volume of each empty space is given in A Additionally the output is in the same format for all three types of elements namely Cavity Void and Molecular surface Last but not least please note that the descriptor Depth is purely a geometrical concept without biological relevance 19 4 6 Refined detection of cavities tunnels and pores In most cases MOLE 2 0 provides relevant results in a fully automated manner without user intervention or the need to understand the algorithms involved in computing empty spaces in biomolecules Nevertheless having even a
4. 1 bd Bond bd index la a 1 model bond append bd cmd set surface_mode 1 cmd set mesh_mode 1 cmd load_model model Tunnels1 cmd hide everything Tunnels1 cmd set surface_color pink Tunnels1 cmd show surface Tunnels1 Tunnels1 def PdbLoadCommand pdbCode 1TQN pdbCode pdbCode upper cmd fetch pdbCode PdbLoadCommand The properties of the identified tunnels as discussed in chapter 4 4 can be exported in XML format MOLE 2 0 will produce an XML file with a content similar to lt xml version 1 0 encoding UTF 8 gt lt Tunnels gt lt Tunnel Cavity 1 Auto 1 Id 1 gt lt Properties Mutability 84 Polarity 13 99 Hydropathy 0 22 Hydrophobicity 0 07 NumNegatives 1 NumPositives 2 Charge 1 gt lt Profile gt lt Node Z 8 660635 Y 0 096446 X 2 175627 Distance 0 000000 T 0 000000 Radius 1 325194 gt lt Node Z 8 701494 Y 0 118397 X 2 169900 Distance 0 046734 T 0 005405 Radius 1 284251 gt lt Node Z 8 741024 Y 0 136001 X 2 161753 Distance 0 090767 T 0 010811 Radius 1 247336 gt lt Profile gt lt Layers gt lt ResidueFlow gt 302 PHE A 305 ALA A 508 HEM A 301 ILE A Backbone 302 PHE A Backbone 301 ILE A 119 SER A 212 ARG A 105 ARG A 108 PHE A 119 SER A Backbone 106 ARG A Backbone 120 ILE A Backbone 120 ILE A 107 PRO A Backbone 122 GLU A 107 PRO A 111 VAL A lt ResidueFlow gt lt LayerWeightedProper
5. Database You may use this point as a potential 23 Figure 25 By tuning the Tunnel parameters on the Refinement panel you can apply filters on which tunnels will be reported A Default parameters allow you to see 3 tun nels leading out of a given caivty B By de creasing the Bottleneck radius parameter a fourth tunnel becomes visible but note that this tunnel has a narrow segment towards its exit C The fourth tunnel can be made visi ble also by increasing the tolerance parameter Bottleneck length D Tightening the sim ilarity criterion Cutoff ratio from 0 9 to 0 5 removes two of the tunnels on the grounds that they share at least 50 of their length with one of the remaining tunnels tunnel start point Simply click on the pink ball in the viewer or tick the corresponding Active box in the Start Points list One or more tunnels may be generated or you may receive an error message in the Status bar depending on whether the chosen point is a relevant tunnel start point Alternatively you may click on the definition of this point in the CSA database section of the Refinement panel This will cause the corresponding amino acid residues to be selected and the pink ball will to be displayed in green as any other user defined start point Then click Compute tunnels or click on the ball to see if any tunnel is found at that position You may also wish to test as many tunnel exit points as possible In order to do so decrease the
6. List of adjustable parameters in MOLE 2 0 How to read parameters In the next page you can find a short description of all parameters for customizing MOLE calculation For a short description of different use cases and structure of XML input file please refer to chapter 6 This overview is particularly useful for those who are working with the command line CL version of MOLE A brief introduction on how to create your own XML input file can be found in chapter 6 Some parameters are more important than others Those of the greatest influence are marked with In case you would like to customize your calculation or the provided output is not in particular what you wanted by tampering these parameters results can be greatly enhanced Except for the input molecule and working directory in CL version there are no compulsory parameters therefore if the argument is not provided a default value is used Please note that default values are hardly sufficient for all different types of calcu lation especially in larger structures gt 500 AA Each row contains a description of a single parameter separated in the columns which can be customized in either one of MOLE versions In columns from left to right you can find the following information Parameter name an attribute name one needs to use in CL version placement of such tag version of MOLE where it can be used data type of the parameter double integer boolean etc default value
7. algorithms for a complete discussion on tunnel starting points please see chapter 4 6 Note that whenever you hover over a residue in the viewer that residue becomes highlighted in yellow and its name and number are displayed at the bottom right corner of the viewer Figure 9 Molecular viewer of the MOLE 2 0 GUI At the top of the molecular viewer there are various options for visualization Figure 10 The Display menu contains controls for the type of molecular structure representation e g cartoon sticks coloring scheme e g by atom by residue etc and background color The Display menu also allows to visualize non protein atoms such as ligands cofactors or solvent molecules Additionally the Display menu enables the representation of cavities as solid volumes and the representation of tunnels as volumes or traces The Start Points menu controls which types of tunnel start points will be displayed for a complete discussion on tunnel starting points please see chapter 4 6 Additional visualization options that can be found at the top of the molecular viewer include the toggle for the display of molecular surface and a clipping plane tool with a scroll bar which allows to explore three dimensional structures in steps Take some time to explore all visualization options 4 3 3 Sequence browser Right under the in built viewer there is a sequence browser Figure 11 which contains the pri mary sequence of the loaded struct
8. an error message is displayed in the Status bar and None can be used to easily show or hide all graphical elements of a certain type The button Clear completely removes all graphical elements of a certain type from the results Cavities are displayed in the viewer as transparent volumes unless the Solid cavities visualization option is marked at the top of the viewer Note that by default only the three largest cavities are selected and displayed in the viewer and if you would like to display other 12 cavities you need to tick the appropriate box on their left Interior cavities are simply cavities which do not communicate with the surface of the protein and thus MOLE 2 0 does not look for tunnels in interior cavities Tunnels and pores can be automatically generated using the Auto button Note that tunnels can also be made visible by directly clicking on their start point colorful ball inside the molecular viewer or by ticking the Active box of their start point on the Refinement panel Only tunnels starting from active start points will be available in the list of Tunnels on the Results panel is Specific Point Residuets CSA Database 2 Figure 16 The Results panel contains the elements that can be displayed in the viewer cavities tunnels pores The additional section Paths allows for the detection of channels between two given points on the protein surface Such points can be specified as coordinates in the 3D space
9. charge along with the average hydropathy hydrophobicity polarity and mutability are given You can also find here the values computed per layer and then averaged over the whole tunnel while weighting according to the length of each layer Tunnel 2 in Specific Point Residue s CSA Database 2 Profile Lining and Properties Start Points 15 Clear Physicochemical properties of lining side chains Results Charge 1 2 1 Hydropathy 0 36 Hydrophobicity 0 13 Polarity 15 29 Mutability 89 00 Layer weighted Physicochemical properties of lining side chains Hydropathy 1 10 Hydrophobicity 0 25 Polarity 20 08 Mutability 92 00 Depth 20 Volume 3589 53 Depth 11 Volume 369 15 eFI SRTATALSLEQKE Rad E E 7 a 18 3 Depth 10 Volume 325 45 31 E o zi Depth 16 Volume 297 17 29 5 0 Depth 10 Volume 296 12 1 24 0 2 0 A Depth 10 Volume 257 50 73 96 o Depth 9 Volume 248 02 reed E o 89 o Depth 10 Volume 247 88 1TQN Tunnel 2 in cavity 1 Copy Profile Lining Props PDB XML Y Cavities 14 None All Sy s s E 6 Depth 9 Volume 240 58 x Depth 12 Volume 211 21 Depth 11 Volume 181 07 Depth 9 Volume 158 69 3 Depth 9 Volume 133 17 Depth 10 Volume 118 58 Interior Cavities 6 None All Tunnels 9 Auto Clear None All Y 1 Length 23 16 Cavity 1 Details Length 27 49 Eaoy Y Details 3 Length 29 96 Cavity 1 Details Length 7 65 esy 2 Details NENNNNNNNNNNN
10. found in protein structures Working directory WorkingDirectory element CL String output Path to the results folder Controls the type of the tunnel start points used If Origin Origin element CL Boolean 1 1 then automatically detected start points are used If 0 other start points must be defined 83 Filtering parameters Parameter Version Type Default value Function Name CL Tag XML placement Bottleneck radius BottleneckRadius Params attr GUI CL Double 1 25 ee e Does E Maximum length of a valid tunnel for which the Bottleneck length BottleneckLength Params attr GUI CL Double 3 0 a aes eaten tee Cutoff ratio MaxTunnelSimilarity Params attr GUI CL Double 0 7 Mr deeree Uan between wwe Anel before one tunnel is discarded 6 Export parameters Parameter Version Type Default value Function Name CL Tag XML placement Controls storing information about the mesh of Mesh Mesh Export attr CL Boolean 0 detected tunnels for subsequent visualization in PyMol MeshGz MeshGz EA CL ROSSO at ee storing of mesh information in a zip file Level of detail of mesh surface The higher the Mesh density MeshDensity Export attr CL Double 1 33 Mesh Density the lower the level of detail in the visualization Cavities export Cavities Export attr CL Boolean 1 Controls whether cavities will be exported Controls whether a PyMol script will be generated PyMOL export PyMol Export attr CL Boolean 1 for subs
11. general impression of the basic steps can help in tailoring the functionality of the program to specific cases and thus enhancing the research experience Therefore we first give a brief overview of the procedure of computing empty spaces in proteins as well as the definitions of the most important concepts We then explain how to use these concepts to refine your calculation along with examples to show how proper tweaking of MOLE 2 0 parameters can improve your results Remember that a great advantage of MOLE 2 0 is the fact that it allows you to interactively tweak the tunnel detection algorithm such that the results are immediately available for inspection and comparison 4 6 1 Overview of algorithm and concepts As soon as the structure of a biomolecule is loaded its atomic structure is represented by Van der Waals spheres centered on the atoms Further the atomic structure is translated into a molecular graph representation which allows for the computation of the molecular surface and its features in a straightforward manner Figure 21 i ii The molecular surface is approximated as the surface accessible to a sphere the size of which is a parameter Probe Radius which can be adjusted in order to control the level of detail provided by the molecular surface Subsequently it is possible to identify cavities on the molecular surface Figure 21 iii A particular concave feature of the molecular surface can be considered a cavity if its width
12. points i e red arrows are the exit points of actual tunnels The Find button limits the search to exit points of already identified tunnels and it is by this algorithm that you are likely to obtain relevant results The User algorithm allows you to define exit points by CTRL click on the molecular surface in the viewer Make sure to first display the molecular surface by ticking the Surface box at the top of the viewer It is possible to filter out uninteresting pores by adjusting the same parameters as for tunnels 25 Part II MOLE 2 0 a command line based application 5 Technical details 5 1 Availability The command line based version of MOLE 2 0 is available free of charge on our website at http mole chemi muni cz 5 2 System requirements The command line version of MOLE 2 0 can be run on Windows Mac OS or Linux Windows users will need the NET framework 4 0 or above meaning that MOLE 2 0 will run on any Windows operating system from Windows XP Service Pack 3 and higher or Windows Server 2003 Service Pack 2 and higher Linux and Mac OS users need to install the Mono running environment http www mono project com version X or higher In case you are using a Debian based distribution of Linux Debian Ubuntu the package mono complete is more suitable than mono runtime 5 3 Installation It is not necessary to install MOLE 2 0 as executable files are provided upon download Simply download the zip arch
13. value of the Surface Cover Radius parameter in the Tunnel Parameters section on the Refinement panel Nonetheless remember that tunnel exit points are displayed in the molecular viewer as red arrows So once you have identified the optimal values for the other parameters it is better to revert to a smaller Surface Cover Radius for better visualization Figure 26 Example of op timal pore computation with the MOLE 2 0 GUI A Using default param eters the location of the pore is found but the pro file of the pore is not re alistic B Increasing the parameter Probe radius from 3 to 9 allows for a smoother and bulkier molecular surface which leads to a proper identifi cation and description of the pore The Tunnel Parameters section provides the opportunity to filter out tunnels based not only on their start and end points but also on their properties Figure 25 The parameter Bottleneck Radius defines the minimum radius of the tunnel at any point along its length Setting Bottleneck Radius a lower value will return more tunnels and its effect is somewhat similar to that of setting Interior Threshold in the Cavity Parameter section Tunnels 24 which are narrower than twice the Bottleneck Radius at any point will not be reported if the parameter Bottleneck Length is set to 0 Nonetheless if you would like to allow for some tolerance you may ask MOLE 2 0 to report also tunnels which are narrower than twice the Bottleneck Rad
14. 0 calculation is done In this case check that the attribute PyMOL in the Export tag of the XML file be set to 1 In this case MOLE 2 0 will generate a PyMOL script which you can then run in PyMOL for more details please see chapters 6 7 and 6 8 below All exported properties can be analyzed as described chapter 4 4 of Part I in this manual 6 6 Refined detection of cavities tunnels and pores As previously mentioned adjusting the way cavities are detected directly affects the tunnels that MOLE 2 0 will be able to identify in a structure Please read through chapter 4 6 of Part I in this manual before proceeding It is possible to restrict the area of the protein where the cavity search will be performed by employing the SpecificChains attribute in the Input tag For example if you would like to include only chains A B and C of the structure from the file 1JJ2 pdb then your Input tag looks like lt Input SpecificChains ABC gt 1JJ2 pdb lt Input gt Note that the attribute SpecificChains is not mandatory The additional tag NonActiveResidues allows for further specification regarding which areas of the structure will be excluded from the calculation The space previously occupied by the excluded residues will be considered as empty which may be useful for instance in studying the protein even if the ligand is present in the cavity or tunnel lt NonActiveResidues gt lt Residue Chain A SequenceNumber 508 gt lt NonAct
15. CBR MU amp FCH UPOL Z v 13 8 28 NCBR MU amp FCH UPOL C amp NCBR MU 8 FCH UPOL role chemi muni cz DO 1Ni CZ 13 8 28 wwy emi muni cz v 13 8 28 Figure 28 The MOLE 2 0 plug in for PyMOL allows you to run MOLE 2 0 calculations from inside PyMOL Basic computation settings can be found in the tabs Compute tunnels and Compute pores Fine tuning can be achieved by adjusting the parameters in the Settings tab Last but not least previous calculations of MOLE can be directly loaded in PyMOL in the Read Channels tab Note that it takes a bit of time to get used to working with PyMOL choosing different visualization modes making selections etc Therefore if you are a first time PyMOL user it is advised that you spend a bit of time exploring the works of PyMOL itself before you attempt to visualize MOLE 2 0 output 6 8 Interactive tunnel calculation with MOLE 2 0 inside PyMOL If you are using Mac OS or Linux you cannot run the GUI based version of MOLE 2 0 Nev ertheless by using our PyMOL plug in you have the opportunity to interactively refine your MOLE 2 0 calculation inside PyMOL in a similar manner as you would inside the MOLE 2 0 GUI If you have already downloaded and unpacked the command line version of MOLE 2 0 simply download the MOLE 2 0 PyMOL plug in from our website at http mole chemi muni cz Then start PyMOL go to Plugins gt Plugin Manager gt Install new plug in navigate to the location where you have down
16. NEB LengtkeT71 Cavity 5 Details Length 11 58 Cavity 6 Details Length 6 83 Cavity 8 Dota Figure 19 The Lining and Properties tab of the properties window needs to be enlarged to be able to display all information Each line in the table contains the description of a layer in the tunnel Further the lining residues of the tunnel are given as a list of their one letter codes in the order in which they contribute to the tunnel surface starting from the bottom of the cavity 16 tunnel starting point and expanding towards the surface of the protein tunnel exit point The tunnel is characterized layer by layer Remember that a tunnel layer is defined by the residues lining it Therefore the order of residues in this list is not given by the amino acid sequence directly but by the overall three dimensional arrangement of the entire protein structure and how the residues come together from distant parts of the sequence to form the surface of the tunnel Hover over any of the residues to get complete information regarding its position in the sequence The residues which contribute to the surface of the tunnel by their backbone part are marked with a dot next to their one letter code Note that if a residue contributes with both its side chain and its backbone it appears twice in the list of residues Each line in the table describes a layer of the tunnel and it contains the list of residues contributing to its surface along w
17. Origin attributel value gt value lt Input gt lt Params attributel value gt lt Export attributel value gt lt NonActiveResidues gt lt NonActiveResidues gt lt CustomVdw gt lt CustomVdw gt lt Tunnels gt 29 The principles of using parameters in refining MOLE 2 0 calculations can be found in Part I whereas a description of all parameters that can be used with MOLE 2 0 is given in Part III To help you along we have included a few use cases in section 6 6 and we provide a couple of test xml input files inside the zip that you download in order to get your command line version of MOLE 2 0 6 2 Loading structures The Input tag allows to specify the path to an input PDB file If your structure of interest is in the file 1HTQ pdb and this file is at the same place as the location from where you are running MOLE 2 0 your Input tag should look like lt Input gt 1HTQ pdb lt Input gt Remember that by default if a PDB file contains more models MOLE 2 0 will use only the first one in the calculation However if you are working with extremely large biological complexes over 100000 atoms various parts of the structure will be stored in the PDB file as separate models pdb1 and pdb2 formats The biologically active unit consists of the atoms of all models You will of course wish to work with the entire biological unit and not only with the part stored in the PDB file as the first model For such situations yo
18. S MOLE 2 0 User manual L CEITEC Central European Institute of Technology BRNO CZECH REPUBLIC Contents 1 Statement of purpose 1 1 Citing MOLE 2 0 2 tots akc AA 2 How to use this manual Part I MOLE 2 0 a Graphical User Interface based application 3 Technical details S31 Availability ceca a Ge kee ae be AA a a 3 2 System requirements 2 e Bio nstalationss es cna 2 ee Bg eee ee Bes a te Bh Si a ie ks 4 Functionality 4 Loading 4 Structure osteo ae a Soe 4 2 Automatic detection of cavities tunnels and pores 4 3 Nisualizing results sis snoa ace a i A ek a hee we o Get Se e AS tls MEA A ass gla oe a wal Dr ess Fe I eee che pie EGR i 4 3 2 Molecular viewer 2 2 aaa ee 4 3 3 Sequence browser oaoa ee 4 3 4 Refinement panel 2 2 2 02 000 e 413 0 Results pan ls m ani 2400 Shite a a Soe Soh ast See Bec E a 43 07 Status bar ee moto ON ete dad A A Od ola 4 4 Interpreting results isa fo eke ee kee Eh he I OE EO i AAW Chargen AN Add WA ydropathiy ae iret amp is a He Mise Se ee ea a at Oo le Sed 4413 Hydrophobicity ysa a a de Rl Ba AAA Polarity tetas rece A a ES Shs REE A i BI A AAS Mita bli A Be aL ee a A a ee od 4 46 Details window 2 44665 sa eoii a aa ne Bea aah aok e deng Gold ans 4 5 Exporting results erraitan i op GS ae Go A AME Soy AD Bog Ghee ete i 4 6 Refined detection of cavities tunnels and pores o ooo o a 4 6 1 Overview of algorithm and co
19. Tunnels Clear Y Specific Point Residue s Point 1 12 0 3 2 Residues LYS 123 A GLU 123 Y CSA Database 2 GLU 308 A THR 309 A THR 309 A PHE 435 A CYS 442 A Start Points 15 Figure 12 Refinement panel of the MOLE 2 0 GUI The upper right part of the workspace Figure 12 contains a set of controls that can be used to refine the cavity tunnel and pore detection algorithms We provide here only a brief overview of the purpose of each section A full description of the concepts and procedures can be found in chapter 4 6 In the sections Chains and Active residues you can restrict the areas of the protein where MOLE 2 0 will search for cavities tunnels and pores Figure 13 The automatic algorithm uses the whole structure but you may unselect whole chains or specific residues if you wish and click the corresponding Update button to apply the changes Note that whenever you hover over a chain or residue in the Chains or Active residues lists those elements become highlighted in yellow in the in built molecular viewer The sections Cavity parameters and Tunnel parameters can be used to fine tune the detection algorithm Details on how these parameters affect the algorithm and the results are given in chapter 4 6 The section called Selection displays the residues that you select via the sequence browser or directly inside the molecular viewer and allows to easily clear the whole selection Additionally it allows to recom
20. UI version of MOLE 2 0 the command line application allows user defined tunnel start points within the Origin tag If the attribute Auto is set to 1 then MOLE 2 0 automatically detects tunnel start points at the bottom of the cavities In order to define a start point first set the attribute Auto to 0 Then identify the start point as a residue or group of residues lt Origin Auto 0 gt lt Residue Chain A SequenceNumber 308 gt lt Residue Chain A SequenceNumber 309 gt lt Origin gt or as a point in the three dimensional space defined by its Cartesian coordinates lt Origin Auto 0 gt lt Point X 1 01 Y 2 35 Z 15 4 gt lt Origin gt Remember that all user defined starting points will be optimized according to the Origin Radius parameter If you do not want such an optimization to take place you can use the ExactPoint tag lt Origin Auto 0 gt lt ExactPoint X 1 01 Y 2 35 Z 15 4 gt lt Origin gt Multiple starting points can be delivered to MOLE 2 0 in a single calculation In this case the Origin tag will contain a separate Pinned tag for each user defined start point lt Origin Auto 0 gt lt Pinned gt lt Residue Chain X SequenceNumber 123 gt lt Pinned gt lt Pinned gt lt Point X 1 Y 2 Z 3 gt lt Pinned gt lt Origin gt 6 7 Visualization in PyMOL While the command line version of MOLE 2 0 does not allow for immediate visual inspection of the results it
21. acid 1 04 for aspartic acid The 15 hydrophobicity of the tunnel or tunnel layer is calculated as the average of the Hdph indices of all lining amino acid residues 4 4 4 Polarity The polarity index Pol is a way to quantify the displacement of positive and negative charges inside a residue Large residues with charged side chains have higher polarity while small neutral residues have minimal polarity The polarity of the tunnel or tunnel layer is calculated as the average of the Pol indices of all lining amino acid residues 4 4 5 Mutability The mutability index Mut of an amino acid residue relates to the probability of that par ticular amino acid mutating into another amino acid High Mut values indicate relatively high propensity for mutation while low Mut values indicate lower propensity for mutation The mutability of the tunnel or tunnel layer is calculated as the average of the Mut indices of all lining amino acid residues which contribute to the tunnel surface with their side chains 4 4 6 Details window The Lining and Properties tab is organized in a table Please note that when the physicochemical properties window first opens it is not large enough to display all the data it contains in the Lining and Properties tab You therefore have to enlarge the window in order to see all the data in the table Figure 19 At the top of the table the values of the properties are given as calculated for the whole tunnel The total
22. cted technique see below you may decide which exit points will be tested when looking for pores The restrictions applied on the exit points are the same as for tunnels It is possible to filter out uninteresting pores by adjusting the same parameters as for tunnels 4 6 2 Refinement techniques The tools needed in this step can be found in the Refinement Panel at the top right corner of the workspace Figure 12 It is important to remember that MOLE 2 0 first identifies cavities and only afterwards looks for tunnels and pores Therefore any operation which affects the way cavities are found and characterized will reflect upon the subsequent tunnels and pores In the sections Chains and Active residues you can restrict the areas of the protein where MOLE 2 0 will search for cavities see also chapter 4 3 4 and Figure 13 After you have made your changes click Update and then recalculate all tunnels and pores by using the corresponding Auto buttons in the Results panel Adjusting the Probe Radius parameter in the Cavity Parameters section allows to refine the level of detail of the molecular surface Figure 22 A lower value of Probe Radius will produce a rough surface following closely on the Van der Waals contour A higher value of Probe Radius will produce a smooth surface with little detail If you use a higher Probe Radius you may find that some tunnels prolong artificially outside the van der Waals area Therefore the default value is u
23. due s section you may specify a point in the three dimensional space by its Cartesian coordinates Al ternatively in the same section you may specify a group of residues the center of which will be used as a potential tunnel starting point Once you have specified some point by coordinates or group of residues click the corresponding Go button in order to generate a user defined start point If this point lies inside some cavity a green ball will appear in the viewer and a new item will be added to the list of Start Points on the Refinement panel marked by the label User Note also that the Selection section is cleared and updated with the specified residues A user start point can be generated also by simply clicking on residues directly inside the viewer but first you will have to clear the selection manually by using the Clear button at the right of the Selection section on the Refinement panel Next click the Compute tunnels button in the Selection section or tick the Active box of the newly added point in the Start Points list or click directly on the green ball in the molecular viewer If any tunnels with that start point are found they will be displayed in the viewer as colorful volumes or traces depending on your display options and added at the top of the Tunnels list in the Results panel If no tunnel is found an error message will appear in the Status bar Do not worry about the accuracy of your specification of the tunnel start
24. e contrary might indicate a pathological mutation or even improper conditions of the simulation Additionally remember that all quantitative evaluations are fairly approximate since they are based on experimental or modeled molecular structures of varying quality 4 5 Exporting results Tunnels Pores Paths To export your results expand the Export section in the Results Tunnels Pores Paths panel Figure 20 and click on the appropriate button The iS graphical representation of tunnels pores and paths expressed as sets of coordinates in the three dimensional space can be Figure 20 You may export exported in PDB format MOLE 2 0 will produce a PDB file your results in the Export XML XML Representation with a content similar to section of the Results panel REMARK ATOM NAM RES TUNID X Y Z Distnm RadiusA HETATM 1 X TUN H 1 17 113 23 724 5 514 0 00 1 33 HETATM 2 X TUN H 1 17 119 23 746 5 473 0 05 1 28 HETATM 706 X TUN H 4 24 207 27 310 32 884 7 55 3 02 HETATM 707 X TUN H 4 24 227 27 318 32 847 7 59 3 02 Moreover MOLE 2 0 allows you to generate PyMOL scripts for comfortable visualization and production of high quality pictures using PyMOL MOLE 2 0 will produce a py file with a content similar to 17 def Tunnelsi model Indexed at Atom at name 0 at vdw 1 3251938904398 at coord 17 113096738422 23 7241725168357 5 51369483241423 model atom append at for a in range len model atom
25. el surface together with the physicochemical properties of the tunnel and its segments Here we must mention that MOLE 2 0 does not define segments as identical units of length but rather as layers Each layer is defined by the residues lining it A new layer starts whenever there is a change in the list of residues lining the tunnel along its length Several physicochemical properties are of interest here Such properties are defined per amino acid residue and can be found in Table 1 In the Lining and Properties tab MOLE 2 0 reports the physicochemical properties per group of residues making up layers of the tunnel or the complete tunnel An overview of the physicochemical properties reported by MOLE 2 0 is 14 Residue Charge Hydropathy Hydrophobicity Polarity Mutability ALA 0 1 8 0 02 0 100 ARG 1 4 5 0 42 52 83 ASN 0 3 5 0 77 3 38 104 ASP 1 3 5 1 04 49 7 86 CYS 0 2 5 0 77 1 48 44 GLU 1 3 5 1 14 49 9 77 GLN 0 3 5 1 1 3 99 84 GLY 0 0 4 0 8 0 50 HIS 0 3 2 0 26 51 6 91 ILE 0 4 5 1 81 0 13 103 LEU 0 3 8 1 14 0 13 54 LYS 1 3 9 0 41 49 5 72 MET 0 1 9 1 1 43 93 PHE 0 2 8 1 35 0 35 51 PRO 0 1 6 0 09 1 58 58 SER 0 0 8 0 97 1 67 117 THR 0 0 7 0 77 1 66 107 TRP 0 0 9 1 71 2 1 25 TYR 0 1 3 1 11 1 61 50 VAL 0 4 2 1 13 0 13 98 Table 1 Physicochemical properties of single amino acid residues given below Note that the properties considered here overlap in physical meaning and are highly correlated e g
26. eq ent visualization in PyMol PyMOL display type PyMolDisplayType Export attr CL String Surface Conte Dane Wau leaion tye one mucny y EEN a y EER k 3 5 de PyMOL Allowed values are Surface and Spheres If this value is present the option for downloading PDB id PDBId Export attr CL String the structure is incorporated in the PyMOL visualization script PDB export PDB Export attr CL Boolean 0 Controls if PDB export is created Tunnels Tunnels Erai CL Boolean 0 Short information about tunnels is printed out to the console Short output ShortOutput Export attr CL Boolean 0 Controls if PDB export is created
27. es can be found in chapter 4 3 4 2 Automatic detection of cavities tunnels and pores In most cases MOLE 2 0 provides relevant results in a fully automated manner without user intervention or the need to understand the algorithms involved in computing empty spaces in biomolecules Simply loading a structure will trigger the automatic detection procedure Fig ure 6 and the results will be available for visualization in several seconds Once the structure is loaded you can simply click the Auto buttons next to Tunnels and Pores on the right hand side of the screen and you will be able to instantly access the results as described below 4 3 Visualizing results First it is important to mention that MOLE 2 0 uses the concept of workspaces to define the visible area of the screen where results are displayed along with all the loaded structures the results themselves and various action buttons Figure 7 Any state of the workspace can be saved into a special file wtw and used at a later time The wtw file stores all your results parameter settings and even camera position so that you can resume your work with MOLE 2 0 at any time The area of the workspace can be divided into several main parts namely the menu in built molecular viewer residue selection bar refinement panel results panel and status bar Figure 7 Workspace of the MOLE 2 0 GUI 4 3 1 Menu The topmost part of the workspace contains the MOLE 2 0 menu Fi
28. f the tunnel start and end points are user defined MOLE 2 0 will find tunnels only if the tunnel start point lies inside some cavity and the exit point lies at the boundary of some cavity Once the tunnel start and end points have been identified the tunnel itself is computed as the shortest distance between the two points on the molecular graph representation Figure 21 vi The visual representation of the tunnel is a surface volume between the start and end point The thickness of the tunnel at each point corresponds to the empty space between the surrounding Van der Waals spheres which represent the atoms of the amino acid residues lining the tunnel Many tunnels are generated by the above described procedure but not all might be rele vant It is possible to filter out tunnels that are not of interest by setting a few parameters Figure 21 vii In order to ensure that the reported tunnel is wide enough for ligands of interest to pass through the minimum requirements for the bottlenecks can be set by specific parameters Bottleneck Radius Bottleneck Length If two identified tunnels follow the same channel leading out of a cavity in too similar a manner only the shorter tunnel will be reported The critical level of similarity can also be adjusted by a parameter Cutoff Ratio It is also possible to identify pores in the protein structure and in principle it is done by finding connections between two tunnel exit points Depending on the sele
29. ght wish to perform using MOLE 2 0 For a full description of the algorithms any questions you may have which cannot be answered by the present material or any suggestions on how to improve MOLE 2 0 please refer to our web pages at http mole chemi muni cz Enjoy working with MOLE 2 0 Part I MOLE 2 0 a Graphical User Interface based application 3 Technical details 3 1 Availability The MOLE 2 0 GUI based application is available free of charge on our website at http mole chemi muni cz 3 2 System requirements The GUI based version of MOLE 2 0 requires the Windows environment More specifically it requires the NET framework 4 0 or above This means that MOLE 2 0 can run on any Windows operating system from Windows XP Service Pack 3 and higher or Windows Server 2003 Service Pack 2 and higher 3 3 Instalation It is not necessary to install MOLE 2 0 as executable files are provided upon download Simply download the zip archive from our website extract to a preferred location on your disk and you can immediately start using MOLE 2 0 by running the executable file mole exe Figures 2 4 EEEE oowoo E R R S S a r E E yebchem ncor muni cz Platforr oles 3 Google MOLE 2 0 OLE 2 0 is an universal toolkit for rapid location and characterization of channels tunnels and pores in bio macromolecular structures Downloads and Links MOLE 2 0 Other MOLE Products Documentation The doc
30. gure 8 which allows to perform general operations such as loading or downloading structures and saving or loading workspaces If more structures are loaded into the MOLE 2 0 GUI based version each of them will appear in its own tab and you can work with each structure completely independently 4 3 2 Molecular viewer The largest part of the workspace contains the in built molecular viewer and buttons related to visualization options Figure 9 Here you can visualize various graphical elements such as loaded MI MOLE 2 13 18 Load 1f4w Download Workspace Load Save 1f4w 1TQN x Figure 8 The menu of MOLE 2 0 GUI contains commands to load or download structures load or save workspaces and switch between different structure tabs structures or any cavities tunnels and pores that MOLE 2 0 has found Additional graphical elements include the molecular surface along with tunnel start points as balls with arrows and tunnel exit points as tetrahedrons with arrows At the bottom left corner of the viewer you can find a quick help box regarding the usage of the mouse inside the viewer select rotate zoom etc Do not forget to look for the tool tips whenever you feel uncertain The built in molecular viewer also allows you to select residues amino acids ligands solvent by clicking on them directly inside the viewer Such selections are useful for differentiated graph ical representation but also to refine the tunnel detection
31. here are a few additional parameters involved in operating with the command line version of MOLE 2 0 Nonetheless tunnel calculation can still proceed in an automatic or refined fashion according to your preference The input file contains XML tags which function as categories or settings for your calculation using MOLE 2 0 Depending on how you set the tags you may run the automatic or refined tunnel detection algorithms and you may decide about which data is exported and how You do not need to adjust or even understand all the parameters included in the XML input file The most important parameters are discussed in the subsections below while the rest are briefly described in the Annex of this manual 6 1 Structure of input XML file The input XML file describes the setup of the ensuing calculation namely it tells MOLE 2 0 which files to use The structure of the input file follows XML standard with the Tunnels element as the root tag In the first level you can find additional elements for specifying the input structure working directory start of the calculation parameters of the calculation and finally parameters related to exporting the results Input WorkingDirectory Origin Params and Export The simplified structure of the input file is as follows lt xml version 1 0 encoding UTF 8 gt lt Tunnels gt lt Input attributel value gt value lt Input gt lt WorkingDirectory attributel value gt value lt Input gt lt
32. is larger than some minimum value Interior Radius As a consequence this parameter basically controls the definition of cavities Figure 21 Overview of the tunnel calculation procedure i ii building the molecular surface iii identifying cavities iv identifying potential tunnel start points v identifying potential tunnel exit points vi computing tunnels vii filtering tunnels Further MOLE 2 0 looks for tunnels which lead from the identified cavities to the surface of the protein The calculation of tunnels as paths between two points e g between the bottom of a cavity to the protein surface requires that the start and end points be defined prior to the calculation Figure 21 iv v Tunnel start points can be assigned automatically at the bottom of each identified cavity or they can be user defined The user may specify a point in the three 20 dimensional space or group of amino acid residues Tunnel start points will be tested within a certain distance around this point This distance is controlled by an adjustable parameter Origin Radius allowing an effective exploration of an area of interest Tunnel exit points can be automatically assigned at the outer boundary of each cavity or can be user defined Many exit points are generated and tested at each cavity boundary The density of exit points that will be tested at cavity boundaries can be controlled by an adjustable parameter Surface Cover Radius Note that even i
33. ith a few descriptors The first descriptor is Rad the minimum tunnel radius along that particular layer Remember that the thickness of the tunnel is twice the value of Rad The second descriptor is Dist the distance from the bottom of the cavity tunnel starting point to the farthest atom in that particular layer The third descriptor is Hdry the average hydropathy index of the group of residues making up the surface of the tunnel layer The fourth descriptor is Hdph the average hydrophobicity index of the group of residues making up the surface of the layer The fifth descriptor is Pol the average polarity index of the group of residues making up the surface of the layer The last index is Mut the average mutability index of the group of residues making up the surface of the layer Note also that the shade of the background of each line in the table indicates whether there is a bottleneck in the tunnel in that particular layer Darker shades indicate local bottlenecks while black indicates the presence of a global bottleneck It is important to note that such results represent static pictures of the identified cavities tunnels and pores During protein dynamics the shape profile might be modified the side chain of some amino acid residues might change position or a tunnel leading out from a cavity might open or close completely Depending on the case such changes might be associated with the protein performing its biological function or on th
34. ius over a length of maximum Bottleneck Length Additionally it is possible to minimize redundancy by filtering out tunnels that are too similar If two tunnels are too similar over a proportion of their length higher than Cutoff Ratio only the shorter tunnel will be reported If Cutoff Ratio is set closer to 1 the similarity criterion is weak and more tunnels will be reported If Cutoff Ratio is set closer to 0 5 the similarity criterion is strong and fewer tunnels will be reported Pores can be computed and displayed only after the tunnels have been identified The reason is that pores are defined as channels which connect two tunnel exit points All parameters which affect tunnels thus affect pores in the same way Figure 26 Moreover special importance is given here to exit points We have already seen that exit points can be defined only at the outer boundaries of cavities Therefore parameters which affect the molecular surface Chains Active residues Probe radius the definition of cavities Interior threshold or the density of possible exit points at the cavity boundary Surface Cover Radius will influence the detection of pore exit points Additionally MOLE 2 0 provides several algorithms which allow a more specific selection of exit points to be tested Figure 16 The Auto button computes the pores as channels between all pairs of potential exit points automatically generated at all cavity boundaries Note that not all potential exit
35. ive from our website Figure 27 extract to a preferred location on your disk and you can immediately start using MOLE 2 0 by running the executable file mole2 exe This executable file takes as input a single XML file which includes all information about the molecular structures of interest the parameters of the calculation and export options for the results IE Scie 20 we LE a a VIE ec 48 weveremncor muniea Patiorn Aoo wea tas v e BF 6000 2 t D MOLE 2 0 a took for rapid location and characterization ol channel tunnels end Downloads and Links MOLE 2 0 O MOLE 2 0 O MOLE 2 0 Gr le Tool Other MOLE Products Documentation The documentation is included with the downloads in PDF format Atematively visit the MOLE wiki page References Figure 27 Download the command line version of MOLE 2 0 from our web page In order to run the command line version of MOLE 2 0 on Windows you need to open a command line prompt then type inside the path to the executable and the path to the xml file as below path to mole2 exe path to xml xml 27 For example if you have already navigated to the folder where you extracted all necessary files of MOLE 2 0 and you have copied in this folder also the xml input file called input xml as well as the molecule you would like to work with you need simply run mole2 exe input xml On any non Windows operating systems with the mono environment insta
36. iveResidues gt Remember that the molecular structure is represented at some point by the van der Waals radii of the atoms Moreover the tunnel size is computed according to the distances between van der Waals spheres The command line version of MOLE 2 0 allows you to customize the van der Waals radii of selected elements via the CustomVdW tag In this case you must provide the van der Waals radii of all elements in the following format 31 lt CustomVdw gt lt Radius Value 1 Element H gt lt CustomVdw gt The Params tag provides ample regulatory control over the calculation This tag is equiva lent to the Refinement panel in the GUI version of MOLE 2 0 see the chapter 4 3 4 and 4 6 from Part I in this manual Additionally the Params tag may contain the RemoveHydrogens attribute lt Params OriginRadius 5 SurfaceCoverRadius 10 InteriorThreshold 1 4 ProbeRadius 5 RemoveHydrogens 1 gt RemoveHydrogens is binary attribute meaning that it can have the value 0 false or 1 true If RemoveHydrogens is set to 1 true all hydrogen atoms in the structure are excluded from the calculation of the molecular surface Note however that excluding the hydrogen atoms will make the structure seem more porous than in reality leading to a higher number of false tunnels that will need to be filtered out Therefore it is advisable to tweak also other parameters in the Params tag when using RemoveHydrogens 1 As in the G
37. le for subsequent visualization in independent molecular viewers Moreover in combination with our PyMOL plug in the command line version of MOLE 2 0 provides interactive functionality comparable to the GUI version under Windows Mac OS and Linux The command line appli cation is ideal for batch processing as it can be included in scripts which process large numbers of structures at a time or which allow for automatic post processing and integration of results 1 1 Citing MOLE 2 0 If you find MOLE 2 0 useful for your work please cite it as e Sehnal D Svobodov Va ekov R Berka K Pravda L Navr tilov V Ban P Ionescu C M Otyepka M Ko a J MOLE 2 0 advanced approach for analysis of biomacromolecular channels Journal of Cheminformatics 2013 5 39 2 How to use this manual All you need to know in order to use MOLE 2 0 effectively is covered by this material The manual discusses the GUI based application first where the majority of concepts procedures and parameters are covered and then the command line based application Note that the part of the manual dedicated to the command line application discusses in detail only those concepts and procedures which are particular to the command line version of MOLE 2 0 Therefore even if you plan to use only the command line application please refer to Part I of the manual for a proper overview of functionality The manual follows the logical flow of operations that a user mi
38. licking on any start point colorful ball di rectly inside the molecular viewer vV 37TRPL v 38VALL v 39GLNL Figure 13 You may exclude part of the structure by unse lecting whole chains or specific residues Note however that sometimes not all start points are visible in the viewer in which case you need to change the visualization options at the top of the viewer to Start Points gt Automatic All 4 3 5 Results panel The lower right part of the workspace Figure 16 contains all graphical elements that can be displayed by the in built molecular viewer such as cavities tunnels and pores If you wish to display one such element simply expand its list and tick the item of interest The buttons All 11 Display Start Points Surface Reset Pan Clipping Plane Hide Controls Refinement gt Chains Active Residues 469 of 469 gt Cavity Parameters Default Tunnel Parameters Default Selection Compute Tunnels Clear Specific Point Residue s gt CSA Database 2 Y Start Points 15 Cavity 2 Computed Cavity 12 Computed Cavity 13 Computed Cavity 3 Computed Cavity 4 Computed Cavity 5 Computed Cavity 6 Computed Cavity 8 Computed Cavity 9 Computed Cavity 10 Computed Figure 14 Potential tunnel start points appear as colorful balls in the viewer Active Residues 469 of 469 Cavity Parameters Default Tunnel Parameters Default Selection Compute Tunnels Clear S
39. lled you just need to open a terminal and run mono path to mole2 exe path to xml xml If you get an error please double check section 5 2 If your system meets all requirements and you still get an error please contact us via our website at http mole chemi muni cz 28 6 Functionality Compared to the GUI based version in the command line version of MOLE 2 0 cavities and tunnels are computed the same way and the same properties can be exported However unlike the GUI application the command line version of MOLE 2 0 does not provide immediate visual ization of the identified cavities tunnels and pores but it does allow for subsequent visualization in PyMOL via automatically generated scripts Moreover in combination with our PyMOL plug in the command line version of MOLE 2 0 provides interactive functionality comparable to the GUI version under Windows Mac OS and Linux The command line version of MOLE 2 0 is ideal for processing a large number of molecular structures at the same time as the call to MOLE 2 0 can be easily be included in scripts The command line version of MOLE 2 0 employs the same basic parameters as the GUI version Therefore please refer to Part I of this manual for details regarding the interpretation of results chapter 4 4 and basic algorithms and concepts chapter 4 6 It is important that you read through Part I of this manual even if you plan to use only the command line version of MOLE 2 0 T
40. loaded the MOLE 2 0 plug in and load this file into PyMOL Now the MOLE 2 0 plug in should be available in the list of available PyMOL plug ins If you do not find it in the list try restarting PyMOL In order to use the MOLE 2 0 plug in simply double click on it in the list A separate window will open where you will be able to setup your MOLE 2 0 calculation Figure 28 At the top of the plug in window there are a few tabs In the Compute tunnels tab you may set the most basic requirements of your calculation Figure 28 A First if you have more than one molecule loaded into PyMOL here you may select which one you will use in your MOLE 2 0 calculation Next you can define potential tunnel start points as a list of residues If you do not specify any start points MOLE 2 0 will automatically generate start points at the bottom of all identified cavities In the Compute tunnels tab you may also specify the location of the output files by the Save output to button 33 Additionally if you would like to get start point suggestions from the CSA database you must specify the location of the CSA database file by the Select CSA dat file button This file was provided to you together with the download of the command line version of MOLE 2 0 Remember that you need to tell PyMOL where to look for the MOLE 2 0 executable file In order to specify the location of the MOLE 2 0 command line application click the MOLE 2 0 location button and navigate to
41. more polar residues are less hydrophobic They are nevertheless presented as separate properties for your convenience 4 4 1 Charge MOLE 2 0 considers that the charge of a residue is given by its protonation state Therefore each amino acid has a formal charge The charge of the tunnel represents the algebraic sum between the charges on positively and negatively charged residues which form the surface of the tunnel However note that residues are included in this count only when their side chains and not their backbones form the surface of the tunnel Such residues are displayed in the list without a dot next to their one letter code 4 4 2 Hydropathy The hydropathy index Hdry quantifies the hydrophobic or hydrophilic character of a residue s side chain Larger values of Hdry indicate higher hydrophobicity e g 4 5 for isoleucine 4 2 for valine while lower values of Hdry indicate higher hydrophylicity e g 4 5 for arginine 3 9 for lysine The hydropathy of the tunnel or tunnel layer is calculated as the average of the Hdry indices of all lining amino acid residues 4 4 3 Hydrophobicity The hydrophobicity index Hdph provides a useful way of evaluating the hydrophobicity of some residues in comparison to others More positive values of Hdph indicate more hydrophobic amino acids e g 1 81 for isoleucine 1 71 for tryptophan while more negative values of Hdph indicate less hydrophobic amino acids e g 1 14 for glutamic
42. ncepts 00 000 4 6 2 Refinement techniques 0 e Part II MOLE 2 0 a command line based application 5 Technical details Dall wAvailabilitys cps asec A Oe es A OA tS Pe end nod 5 2 System requirements 2 2 e Dss Installations rr da ee Bea s Ss ag ol kee as E te ag 27 6 Functionality 29 6 1 Structure of input XML file 0 0 0 20 20 0000000000082 29 6 2 Loading structures iia a ee OA OR A eS ee 30 6 3 Automatic detection of cavities tunnels and pores 30 GA Exporting results imac ararte bata SS oa le Poe GS BE ESE ee 31 6 5 Interpreting results ee 31 6 6 Refined detection of cavities tunnels and pores e 31 6 7 Visualization in PyMOL e 32 6 8 Interactive tunnel calculation with MOLE 2 0 inside PyMOL 33 Part III List of adjustable parameters in MOLE 2 0 35 1 Statement of purpose The empty spaces in a protein or a protein complex can be classified as pockets cavities channels tunnels and pores Figure 1 A pocket is a shallow depression on the molecular surface and often serves as binding site for ligands or other biomolecules A cavity is an empty space buried inside the protein structure Cavities may communicate with the exterior environ ment via channels leading up to the protein surface or may be completely isolated from the exterior environment voids Cavities often constitute enzymatic reaction sites as they p
43. or as the geometrical centre of a group of residues Nevertheless the Paths section is still in beta state and should be used with care Note that whenever you hover over a certain graphical element in any list in the Results panel the element is displayed in the viewer in yellow Also note that whenever cavities are connected to the surface of the protein a green tetrahedron and one or more red arrows are displayed in that area These additional graphical elements are there to mark exit points which are important in detecting tunnels and pores Such elements cannot be operated with in the Results panel but they can be adjusted during the refined detection procedure More details on exit points can be found in chapter 4 6 The Results panel also contains an explicit Export section which will be discussed in detail in chapter 4 5 Each graphical item in the Results panel contains also the values of properties of interest such as depth in A and volume in A of cavities and length in A of tunnels and pores Moreover clicking the Details button opens a new visualization window where a more detailed analysis can be performed for each tunnel see chapter 4 4 4 3 6 Status bar The bottom part of the workspace represents the status bar Figure 17 which gives information regarding the type and duration of all operations performed in MOLE 2 0 The status bar provides a useful log of all calculations with MOLE 2 0 including possible errors
44. parameters which directly define the tunnel and which can be found in the Tunnel parameters section of the Refinement panel Note that making changes to the Tunnel parameters does not require you to recompute the tunnels by using the Auto button The reason is that Tunnel parameters affect simply which tunnels will be reported to you and not which tunnels MOLE 2 0 is able to find We have already mentioned that MOLE 2 0 calculates each tunnel as the path between the tunnel start and exit point and thus imposing restrictions on these points is an easy way to filter out tunnels There are several possibilities for identifying tunnel start points Figure 24 a MOLE 2 0 can automatically detect possible tunnel start points at the bottom of all identified cavities These automatically detected start points appear as small blue spheres in the molecular viewer though they are only visible if their parent cavity is active i e if there is a tick in the Active box of that cavity in the list of cavities on the Results panel Automatically detected start points can be found in the list of Start Points on the Refinement panel and are marked by the label Computed 22 CSA defined centre of active site suggested by the CSA database Figure 24 Potential tunnel start points can be automatically generated user defined or inferred from documented active sites b Tunnel start points can also be completely user defined In the Specific Point Resi
45. pecific Point Residue s CSA Database 2 Start Points 15 Cavity 2 Computed Cavity 12 Computed Cavity 13 Computed Cavity 3 Computed Cavity 4 Computed Cavity 5 Computed Cavity 6 Computed Cavity 8 Computed Cavity 9 Computed Cavity 10 Computed Cavity 11 Computed Cavity 1 Database Cavity 1 Database X Cavity 1 User Y Start Points 15 Cavity 1 Computed Cavity 13 Computed Cavity 3 Computed Cavity 4 Computed Cavity 5 Computed Cavity 6 Computed Cavity 8 Computed Cavity 9 Computed Cavity 10 Computed Cavity 11 Computed Cavity 1 Database Cavity 1 Database X Cavity 1 User Pinned Results Cavities 14 Interior Cavities 6 Tunnels 0 Auto Clear None Pores 0 Find User Auto Clear None Paths 0 beta Clear None Export 147 HSHGLFRKLGIPGPTPLPFLGNILSYHKGFCMFDMECHKKY GKVWGFY DGOQOP PZAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAIS IAEDEEWKRLRSLLS PTF TSGRLKEMVPIIAC lomputed 1 tunnel s from the origin wit puted1 in 0 2s ouid not find any tunnels from the origin with 1D Computedi0 Try changing the computation parameters for example lowering the interior threshold or bottleneck radius Figure 15 A In order to look for any tunnels that start from a specific point make the point active by either ticking the Active box in the list of Start Points or clicking directly on the colorful ball in the viewer B If no tunnel is found
46. point By default MOLE 2 0 will test several start points within a certain radius of your initial specification If you would like to test a wider area of space around your specified point simply increase the value of the parameter Origin Radius in the Tunnel Parameters section of the Refinement panel If no points within Origin Radius of your initial specification lie inside a cavity MOLE 2 0 will not display any green ball in the viewer and will not search for tunnels with such start points If you would like to remove all user defined start points use the Clear button in the Start Points section of the Refinement panel If you want to remove only some not all user defined start points first tick the Pinned box of the points you wish to keep and then hit the Clear button c Additionally MOLE 2 0 can retrieve information from the Catalytic Site Atlas CSA a database of known catalytic sites If you have a working Internet connection and you load your protein with its PDB ID MOLE 2 0 will look up the CSA database for any entries related to this PDB ID If a catalytic site is found in CSA the amino acid residues which make up this catalytic site will be listed as an item in the CSA database section of the Refinement panel and the geometrical centre of the catalytic site will appear in the molecular viewer as a pink ball Moreover an additional item will appear at the bottom of the Start Points list on the Refinement panel marked by the label
47. provides you the possibility to do so using PyMOL a free and versatile molecular visualization package Once you have PyMOL installed on your computer start it Then go 32 to File gt Run and navigate to the directory where your MOLE 2 0 calculation has produced output This is the folder that you have set via the tag WorkingDirectory in your input XML file If you had set the PyMol attribute in the Export tag to 1 you should find a file called tunnels py Load this file into PyMOL and you should be able to immediately see the molecule and tunnels Compute Tunnels Settings Compute Pores Read Channels Quick Guide Authors Specify input structure Specify pores starting points Probe Radius alla La Starting Points al Interior Threshold _ 1 25 Add Starting Points Remove Starting Points Surface Cover Radius 4 10 Specify pores end points End Points Origin Radius dls Add Starting Point Remove Starting Point _ Refresh Structures F Overwriteresuts 7 Poresexport I Ignore HETeroatoms BottleneckRadus 41 25 Add End Points Remove end points Save output to ld temp Compute pores BotteneckLengh 3 Generate CSA sites Select CSA dat fie d work _phd pdb_study mole new_csa dat Cutoff Ratio 0 7 MOLE 2 0 location id uworka_phd mole mole_softmole2_cmd mole Additional settings Compute Tunnels I Read all models ITEC amp N
48. pute tunnels once the settings have been adjusted see chapter 4 6 for details The sections Specific point Residue s and CSA Database contain further controls for tunnel starting points which will be discussed in detail in chapter 4 6 The section Start Points contains a list of all potential tunnel start points generated by the settings in all the above mentioned sections This list isn t actually used to refine the calculation but it is a tool which allows you to closely follow all the changes you achieve during the refinement For now it is important to note that tunnel start points are displayed in the molecular viewer as balls of different colors depending on their type and whether they are active or not Figure 14 Refinement Y Chains VH L Y Active Residues 426 of 426 1GLNL 2 ALAL 3 VALL 4VALL 5 THR L 6 GLNL 7GLUL 8 SER L 9 ALAL 10 LEU L 11 THRL 12 THRL 13 SERL 14 PROL 15 GLY L 16 GLUL 17 THRL 18 VALL Y 19THRL Y 20LEUL Y 21THRL Y 22CYSL Y 23ARGL Y 24SERL Y 25SERL Y 26THRL Y 27GLYL Y 28THRL Y 29VALL Y 30THRL v 31THRL v 32SERL v 33ASNL V 34TYRL V 35ALAL V 36ASNL Update Update Not all start points constitute the origin of relevant tunnels If you tick the Active box of a start point you will either notice that one or more tunnels become displayed in the viewer Fig ure 15 A or that an error message appears in the Status bar under the Sequence browser Figure 15 B You can achieve the same effect by c
49. rka K Hanak O hnal D Ranas_P Navratilova V sIalswal_D Ionescu C M Svobadava Varakova_R Ko a Otvenka Figure 3 Unzip the files at your prefered location e E SS a jabcher fo N 3 muniez attom Aop M eao eoma m m ay a TOME creian TumelsBeta Tumelseta 4 se a Organize E Open Share with Bum New okier E ke Favorites Name E Jate modified Typ ia ka E Destcop 4 AvatonDcka 11 635 9M Application exten a I Recent Places AvalonDockTnemesdll j APM Application exten kE x a Downioads csAdat DAT Fl azae Dropbox SalasoriMwmigrteras wer an Application exter ora 7 alma WPF ax I Libraries He oia a p aiite MicrosoftCompilerServicesAsyncTargeti KE d Mus Microsoft Expression Interactions 0107260M A oke 8 Pictes M moise N52031237A Appia ess Kel Videos A PyMOLBuilder ail 4 25 2012 407 PM Application extens B QuickGraph dil x 10PM Applic atens 40 KB Y Homesroup 2 Systemineractvedt 22 21 2011 237 PM Application exten T System Reactvedt 31 2011 201 PM Application exter A Computer SystemReactveWindows Teasing 12 31 2011 254PM_ Apglication exer 168 wos sytem Windows Controls DataVisualza pletion exe Hl 20 Rom Drive E D2PAK a system Windowslnteractiviy dll Application stens 5 WetChemistrysramework Corean yaya pan a Network WebChemistryFrameworkGeometyall 11 21 2012 808 PM Application extens 716 Webcremisiysiteginderani 1 21 2012808PM Application exe 176KB
50. rkingDirectory gt auto_output lt WorkingDirectory gt lt Params OriginRadius 5 SurfaceCoverRadius 10 InteriorThreshold 1 4 ProbeRadius 3 gt lt Export PDB 1 PyMol 1 MeshDensity 1 33 Cavities 1 MeshGz 1 Mesh 1 gt lt Origin Auto 1 gt lt Tunnels gt 30 6 4 Exporting results The data and format that will be exported is controlled at the level of the WorkingDirectory and Export tags The WorkingDirectory tag should contain the path to the folder directory where the output data are going to be stored after the calculation is done If the folder does not exist MOLE 2 0 will create such a folder All properties of all tunnels are exported by default and the functionality is equivalent to that described in chapter 4 5 of Part I in this manual In order to export the PDB representation of the identified tunnels make sure that the attribute PDB of the Export tag has the value 1 Otherwise set it to 0 To export also the cavities set the Cavities attribute of the Export tag to 1 too Remember that MOLE 2 0 also provides information about the physicochemical properties of the cavities containing tunnels along with voids and molecular surface This information can be found in the cavities xml file generated by MOLE 2 0 6 5 Interpreting results Since immediate visualization cannot be achieved directly by the command line application you may wish to visualize the cavities and tunnels using PyMOL once your MOLE 2
51. rovide a highly controlled environment A channel or tunnel is a ligand accessible pathway leading from the protein surface to the interior of a cavity A pore is considered here as a channel that passes through the biomacromolecule from one point on the surface to another Most pores serve as selective transport pathways across membranes Figure 1 Types of empty spaces in proteins A pockets B cavities C tunnels D pores MOLE 2 0 represents cutting edge software for a rapid detection and physico chemical char acterization of tunnels pores and cavities in biomacromolecules proteins nucleic acids lipids glycans and various complexes with emphasis on proteins and large protein nucleic acids com plexes MOLE 2 0 enables a brand new user experience as it can be used effectively even without knowledge of the underlying algorithms This tool comes in two versions either as a Graphical User Interface GUI based application with an in built molecular viewer or as a standalone command line application The functionality of tunnel calculation and characterization is com mon to both versions of MOLE 2 0 but there are some differences The GUI based application works under Windows and allows the user to immediately visualize the results as well as to re fine the calculation in an interactive manner The command line application which works under Windows Linux and Mac is not interactive but the results can be exported in a form suitab
52. s 14 Y 1 Depth 20 Volume 3589 53 ES lt SS Se P Y 2 Depth 11 Volume 369 15 y la y Depth 10 A 4 a 5 A Depth 16 Depth 10 FELA lt 4 Depth 10 av Depth 9 Profile Lining and Properties 3 4 Depth 10 Volu Ox E 9 Depth 9 Volu p X 10 Depth 12 ss gt s 11 Depth 11 gt NN 7 12 Depth 9 13 Depth 9 Volu 14 Depth 10 Volume 118 58 Interior Cavities 6 None All Tunnels 9 Auto Clear None All X Y L Length 23 16 Cavity 1 Details X Y 2 Length 27 49 Cavity 1 Details X Y 3 Length 29 96 Cavity 1 Details X 4 Length 7 65 Cavity 2 o Figure 18 Click on the Details button to open a window which contains the properties of the tunnel which can be exported in various formats The Profile tab describes the thickness of the tunnel along its length The Profile tab contains a plot of the tunnel thickness along the length of the tunnel The X axis follows the length of the tunnel while the thickness is evaluated and represented on the Y axis as the radius descriptor which is half of the diameter thickness of the tunnel A narrow part of the tunnel or pore meaning an area where the radius is small represents a tunnel bottleneck Both the radius and length are expressed in Such graphs give an idea regarding the landscape of the tunnel or pore and help in predicting how a ligand may proceed through The Lining and Properties tab contains a list of residues lining the tunn
53. s that it contains a lot of tool tips to help you along If you are unsure about the meaning of a button parameter or value simply hover with the mouse cursor over it in order to get a basic explanation regarding that particular item 4 1 Loading a structure Immediately at startup you have the opportunity to load a structure of interest Figure 5 Molecular structures can be loaded into MOLE 2 0 in the RCSB Protein Data Bank format pdb If the pdb file contains more structural models as is the case with structures deter mined by NMR experiments only the first model will be used If you wish to use a different model you need to copy and save only the particular model of interest into a separate file and load it separately into MOLE 2 0 Alternatively MOLE 2 0 is able to find retrieve and load any structure directly from the Protein Data Bank website if you provide it with the PDB ID and you are connected to the Internet Note that you can load and work with more than one structure at a time Structures can be loaded into MOLE 2 0 at any time using the appropriate button more about this in chapter 4 3 PA MOLE 2 13 18 gt o off E S Computing Cavities 33s Figure 6 Automatic detection of cavities tunnels and pores using MOLE 2 0 If you have used MOLE 2 0 before and saved a workspace you may load the saved workspace and all related content including structures at this time A detailed explanation regarding workspac
54. sually suitable for smaller more compact structures while a higher value might be useful in the case of large structures or when looking for tunnels with broad profiles Adjusting the Interior Threshold parameter in the Cavity Parameter section allows to identify all voids wider than double the Interior Threshold Figure 23 A small Interior Threshold will therefore result in the identification of more cavities and probably also more tunnels Sometimes it is useful to see even tunnels which seem too narrow for the ligand of interest if you suspect that the protein moves significantly before or during ligand binding On 21 Figure 22 The parameter Probe radius controls the level of detail of the surface A Probe radius 3 B Probe radius 12 the other hand if the protein contains wide voids using a larger Interior Threshold will make it easier to see only those tunnels which are biologically relevant Figure 23 The parameter Interior threshold controls the minimum definition of cavities A Interior threshold 1 25 B Interior threshold 1 90 Remember after you have made your changes to the Cavity parameters you need to tell MOLE 2 0 to generate and display the new tunnels and pores by using the corresponding Auto buttons in the Results panel Further you may filter out tunnels which you presume will not be biologically relevant or which you are not interested in at that time Tunnel filtering can be achieved by adjusting
55. the appropriate folder where you downloaded and extracted the files from our website You may now start the calculation by using the Compute tunnels button The Settings tab Figure 28 B allows you to refine the calculation in a manner equivalent with the Cavity parameters and Tunnel parameters described in chapters 4 3 4 and 4 6 from Part I in this manual The Compute pores tab gives you precise control over which points are used during the pore calculation procedure for details please see chapter 4 6 Once you have set the appropriate parameters start the MOLE 2 0 calculation via the Com pute tunnels button You will be able to visualize all tunnels inside PyMOL directly and moreover all physicochemical properties will be available in the working directory as described in the chapters 4 4 and 4 5 If you experience any issues in working with the PyMOL plug in please contact us via our website at http mole chemi muni cz Again remember that while the MOLE 2 0 plug in offers lots of tool tips to guide you through setting up your MOLE 2 0 calculation inside PyMOL working with PyMOL itself is not very straightforward for complete beginners Thus if you are a first time PyMOL user it is strongly advised that you spend a bit of time exploring the works of PyMOL itself especially choosing different visualization modes making selections etc before you attempt to run MOLE 2 0 calculations using the MOLE 2 0 PyMOL plug in 34 Part III
56. ties Mutability 87 Polarity 15 51 Hydropathy 0 42 Hydrophobicity 0 08 gt 18 lt Layer LocalMinimum 0 EndDistance 0 22977 StartDistance 0 00000 MinRadius 1 22635 gt lt Residues gt 302 PHE A 305 ALA A 508 HEM A lt Residues gt lt FlowIndices gt 0 1 2 lt FlowIndices gt lt Properties Mutability 75 Polarity 0 18 Hydropathy 2 30 Hydrophobicity 0 69 NumNegatives 0 NumPositives 0 Charge 0 gt lt Layer gt lt Layers gt lt Tunnel gt lt Tunnels gt Another way to export the properties of each tunnel is to use the export property menu at the top of the window containing the physicochemical properties of the respective tunnel Simply click the Details button on the right side of the tunnel of interest in the Results panel and then click the appropriate format at the top right corner of the newly opened window Figure 18 The results will be exported to clipboard in your chosen format and then you may paste them in whichever type of file you prefer MOLE 2 0 also provides information about the physicochemical properties of the cavities containing tunnels The same information is provided for voids and molecular surface This information can be found in the cavities xml file generated by exporting the results using the Export tab in the Results panel The structure of the resulting XML file is as follows lt Cavities Version 2 13 8 2 gt lt Cavity Type Cavity Volume 89448 752 Depth 57 Id 1
57. u nentalion fs included with the downloads in PDF formal Alternatively visit the MOLE wiki page References MOLE 2 Ani Figure 2 Download the GUI version of MOLE 2 0 from our web page After the execution of mole exe the starting screen should appear as seen in Figure 5 If you get an error during startup please double check chapter 3 2 If your system meets all requirements and you still get an error please contact us via our website at http mole chemi muni cz BEER nue 20 ercremisy E SS eS E de anche muni e2 lstform App TO ak 5 I sea a p Organizer JOpen Sharewitn Bum New folder 10 de Favores Type Size E desttop Tunelsber zin 1asamanaama Compressed izipp Recent Places Open I Downloads Open in new window J Dropbox mee Estrato Tumesbeta Doe di Scan with Microsoft Security Essentials Music Open with eis Share with Restore previous versions B Videos Send ta 3 4 Homegroup cut Copy Ae Computer Cony wosa H 0 FOM Drive E D2PAK Erari Rename We Network Properties TunnelsBetazip ate modifies 1 15 2013 222PM Date created 1 16 2013 1 48 PM presse zipped Folder Size 254 Mt MOLE 2 0 Sehnal D Berka K Banas P Navratilova V Pravda L Ionescu C M Svobadova Varekova Koca J Olyepka M MOLE 2 0 Tool for Analysis of Biomolecular Channels in preparation MOLEonline 2 0 Be
58. u may employ the Read AllModels attribute in the Input tag lt Input ReadAllModels 1 gt 1TQN pdb1 lt Input gt ReadAllModels is a binary attribute meaning that it may take the value 0 false or 1 true If ReadAllModels is set to 1 true MOLE 2 0 will then use the atoms in all the models to make up the molecular surface of the entire biological unit and you will be able to search for tunnels throughout Note that the functionality encoded in the ReadAllModels attribute is specifically intended to allow the reconstruction of very large biological assemblies and should be used only for the cases where the parts of the structure stored in different models do not overlap Therefore if you are working with a regular PDB file in which the different models contain different conformations of the same molecule and not different subparts of one molecule ReadAllModels should be set to 0 false If you would like to study more NMR models from the same PDB file you will need to manually extract the other models in separate PDB files and MOLE 2 0 will process them independently 6 3 Automatic detection of cavities tunnels and pores If you would like to run the automatic tunnel detection algorithm just use the file testin put_auto xml provided together with the MOLE 2 0 executables in which you simply adjust the Input tag accordingly lt xml version 1 0 encoding UTF 8 gt lt Tunnels gt lt Input gt 1HTQ pdb lt Input gt lt Wo
59. ure Residues amino acids ligands solvent can be selected Display Start Points Surface Reset Pan Clipping Plane Display Start Points Surface Reset Pan Clipping Plane v Cartoons e v CAS Backbone v From Selection Sticks Automatic v Per Cavity All Solid Cavities N one Coloring gt Background gt W v Spheres Centerline vV HET Atoms Water Atoms Figure 10 Detailed visualization options can be found at the top of the molecular viewer by clicking on their one letter codes in the sequence Such selections are useful for differentiated graphical representation but also to refine the tunnel detection algorithms for a complete dis cussion on refinement techniques please see chapter 4 6 Note that whenever you hover over a residue in the sequence browser that residue becomes highlighted in yellow in the in built molecular viewer A 37 47 57 67 77 87 97 107 117 HSHGLFRKKLGIPGPTPLPFLGNILSYHKGFCMFDMECHKKYGRKVWGFYDGOOPVLAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAISIAEDEEY Figure 11 The sequence browser of the MOLE 2 0 GUI contains the list of residues in the structure 10 4 3 4 Refinement panel Refinement gt Chains Active Residues 469 of 469 v Cavity Parameters Default Probe Radius 3 00 Interior Threshold Y Tunnel Parameters Default Origin Radius 5 00 Surface Cover Radius 10 00 Bottleneck Radius 1 20 Bottleneck Length 0 00 Cutoff Ratio 0 90 Y Selection HEM 508 A Compute
60. used if not provided by a user and finally a brief description of its function 36 LE Input parameters Parameter Version Type Default value Function Name CL Tag XML placement Allows to use the atoms in all structural models Read all models ReadAllModels Input attr CL Boolean 0 present the input pdb pdb1 pdb2 file to build the molecular surface of the entire biological assembly a Allow to select chains where MOLE will look for Specific chains SpecificChains Input attr GUI CL String all chains anad ee A AB ABO El Remove hydrogens RemoveHydrogens Params attr CL Boolean 0 All OMR to Eae o o EARE calculation F Regulates level of detail of the molecular surface Probe radius ProbeRadius Params attr GUI CL Double 3 raoba eel Minimum radius of void inside the protein Interior threshold InteriorT hreshold Params attr GUI CL Double 1 25 structure so that the void would be considered a cavity Origin radius OriginRadius Params attr GUI CL Double 5 Fe ed ea ee uDo GS a search for tunnel start points to a sphere of radius Regulates the density of exit points tested at each Surface cover radius SurfaceCoverRadius Params attr GUI CL Double 10 outer boundary Higher Surface Cover radius produces a lower density of exit points Ignore hetero atoms IgnoreHETAtoms Params attr CL Boolean 0 een iy o calculation Custom vdw radius CustomVdW element CL Double per element o ana e m Pa raono various atoms

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