BioLuminate 1.9. Quick Start Guide. Schrödinger Press

Transcription

1 BioLuminate Quick Start Guide BioLuminate 1.9 Quick Start Guide Schrödinger Press

2 BioLuminate Quick Start Guide Copyright 2015 Schrödinger, LLC. All rights reserved. While care has been taken in the preparation of this publication, Schrödinger assumes no responsibility for errors or omissions, or for damages resulting from the use of the information contained herein. Canvas, CombiGlide, ConfGen, Epik, Glide, Impact, Jaguar, Liaison, LigPrep, Maestro, Phase, Prime, PrimeX, QikProp, QikFit, QikSim, QSite, SiteMap, Strike, and WaterMap are trademarks of Schrödinger, LLC. Schrödinger, BioLuminate, and MacroModel are registered trademarks of Schrödinger, LLC. MCPRO is a trademark of William L. Jorgensen. DESMOND is a trademark of D. E. Shaw Research, LLC. Desmond is used with the permission of D. E. Shaw Research. All rights reserved. This publication may contain the trademarks of other companies. Schrödinger software includes software and libraries provided by third parties. For details of the copyrights, and terms and conditions associated with such included third party software, use your browser to open third_party_legal.html, which is in the docs folder of your Schrödinger software installation. This publication may refer to other third party software not included in or with Schrödinger software ("such other third party software"), and provide links to third party Web sites ("linked sites"). References to such other third party software or linked sites do not constitute an endorsement by Schrödinger, LLC or its affiliates. Use of such other third party software and linked sites may be subject to third party license agreements and fees. Schrödinger, LLC and its affiliates have no responsibility or liability, directly or indirectly, for such other third party software and linked sites, or for damage resulting from the use thereof. Any warranties that we make regarding Schrödinger products and services do not apply to such other third party software or linked sites, or to the interaction between, or interoperability of, Schrödinger products and services and such other third party software. May 2015

3 Contents Document Conventions... v Chapter 1: Introduction... 1 Chapter 2: Using the Toggle Table... 3 Chapter 3: Examining Sequences Chapter 4: Identifying Reactive Residues Chapter 5: Identifying Consensus Waters Chapter 6: Homology Modeling of Proteins Chapter 7: Scanning for Residue Mutations Preparing the Protein Setting Up the Mutations Setting Options and Running the Job Examining the Results Chapter 8: Locating Possible Mutations for Disulfide Bridges Chapter 9: Modeling an Antibody Importing the Structure Filtering the Templates To Be Used Searching for Templates Generating the Basic Loop Models Preparing the X-Ray Structure for Comparison Comparing the Model with the X-Ray Structure Humanizing the Antibody BioLuminate 1.9 Quick Start Guide iii

4 Chapter 10: Protein-Protein Docking General Protein-Protein Docking Docking the Ligand Protein As Is Docking the Prepared Ligand Protein Docking the Native Ligand to the Native Receptor Comparing the Top Poses Calculating the RMSD Between the Top Poses Examining the Poses and the Binding Sites Antibody-Antigen Docking Preparing the Antibody Setting Up and Running the Job Comparing the Best Pose with the Reference Getting Help iv Schrödinger Software Release

5 Document Conventions In addition to the use of italics for names of documents, the font conventions that are used in this document are summarized in the table below. Font Example Use Sans serif Project Table Names of GUI features, such as panels, menus, menu items, buttons, and labels Monospace $SCHRODINGER/maestro File names, directory names, commands, environment variables, command input and output Italic filename Text that the user must replace with a value Sans serif uppercase CTRL+H Keyboard keys Links to other locations in the current document or to other PDF documents are colored like this: Document Conventions. In descriptions of command syntax, the following UNIX conventions are used: braces { } enclose a choice of required items, square brackets [ ] enclose optional items, and the bar symbol separates items in a list from which one item must be chosen. Lines of command syntax that wrap should be interpreted as a single command. File name, path, and environment variable syntax is generally given with the UNIX conventions. To obtain the Windows conventions, replace the forward slash / with the backslash \ in path or directory names, and replace the $ at the beginning of an environment variable with a % at each end. For example, $SCHRODINGER/maestro becomes %SCHRODINGER%\maestro. Keyboard references are given in the Windows convention by default, with Mac equivalents in parentheses, for example CTRL+H ( H). Where Mac equivalents are not given, COMMAND should be read in place of CTRL. The convention CTRL-H is not used. In this document, to type text means to type the required text in the specified location, and to enter text means to type the required text, then press the ENTER key. References to literature sources are given in square brackets, like this: [10]. BioLuminate 1.9 Quick Start Guide v

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7 BioLuminate Quick Start Guide Chapter 1 Chapter 1: Introduction The exercises presented in this manual illustrate the use of BioLuminate for a variety of applications. As well as the steps involved to run the main application, each exercise includes any necessary preparatory steps and steps for analysis of results. The exercises all use structures or sequences from the PDB. Some of them run BLAST searches. You should ensure that you have access to the PDB and BLAST databases, either locally or on the web. Chapter 2 covers basic operations in the BioLuminate interface, focusing on the Toggle Table. Chapter 3 introduces the Multiple Sequence Viewer and some of its functions. Chapter 4 contains an exercise on identifying and assessing reactive residues in a protein. Chapter 5 contains an exercise on visualizing the consensus between homologous proteins for species other than the protein itself in this case, water. Chapter 6 contains an exercise on homology modeling of a small protein, with analysis of the quality of the model using a Ramachandran plot, deviations from ideal values of structural parameters, and visual comparison to the known X-ray structure after alignment of the model and the known structure. Chapter 7 contains an exercise on scanning a protein for residue mutations and assessment of the binding affinity of the two protein chains for each other as a function of the mutation. It includes a full protein preparation. Chapter 8 contains an exercise on scanning a protein for pairs of residues that could be mutated to cysteine to form a disulfide bridge, and ranking of the candidates by their effect on protein stability. Chapter 9 contains an exercise on homology modeling of an antibody, with visual comparison to the known X-ray structure after structural alignment. The exercise also demonstrates use of the Workspace sequence viewer to select chains and residues. Information on getting help is provided at the end of this document. BioLuminate 1.9 Quick Start Guide 1

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9 BioLuminate Quick Start Guide Chapter 2 Chapter 2: Using the Toggle Table The set of exercises in this chapter provides a basic introduction to the use of the BioLuminate interface for displaying structures. The focus is on the Toggle Table panel, which is the primary tool in BioLuminate for interacting with objects in the Workspace. First, a structure must be imported. 1. Choose File Get PDB. The Get PDB File dialog box opens. 2. Enter 2ot3 in the PDB ID text box, and click Download. An information box is displayed, indicating that there are problems in the structure. This often means that there are atoms missing, or nonstandard residues. It is an indication that some action to fix the structural issues must be taken before using the structure for modeling. This exercise does not involve any modeling, so the warning is of no concern here. If you don t want to see this message again check the Do not show this dialog again box. 3. Click OK in the Information box. The 2ot3 structure is imported into the project and displayed in the Workspace. The bonds are represented as lines. The color scheme indicates what kinds of problems were found when importing the structure see Section of the Maestro User Manual for more information. These structural defects are of no concern for this exercise, but if you want to use the structure for modeling, these defects would need to be fixed. Later chapters include exercises in which the defects are fixed in the Protein Preparation Wizard panel. Now that the structure is imported, you can start with some basic operations on the view. 4. Rotate the structure in the Workspace: If you have a mouse, hold down the left mouse button and drag horizontally. If you have a trackpad, hold down CTRL ( ) and click and drag on the trackpad. If you have not configured your trackpad, go to Edit Settings Mouse Actions and choose Customize mouse actions for Trackpad. As the structure rotates, parts of it become lighter in shade and parts become darker. This is a 3D effect called fogging that helps distinguish closer atoms from more distant atoms. BioLuminate 1.9 Quick Start Guide 3

10 Chapter 2: Using the Toggle Table 5. Click Reset in the Toggle Table panel. The view of the structure is reset to the original view. 6. Zoom in on the structure: If you have a mouse, hold down the right button and drag. If you have a trackpad, use the pinch gesture or a two-finger swipe. 7. Click Zoom in the Toggle Table panel. The view of the structure zooms out so you can see all the visible atoms. 8. Click Orient. The structure is reoriented so that it is centered in the Workspace, and the view is rotated so that the structure is most spread out in the plane of the screen (the xy plane). Note: This action changes the coordinates of the structure, so you should only use it when changing the coordinates does not matter. Figure 2.1. The BioLuminate main window with the oriented 2OT3 structure. 4 Schrödinger Software Release

11 Chapter 2: Using the Toggle Table In the Toggle Table, there are two rows under Entries in the Workspace, labeled All, and 2OT3. Each row corresponds to a project entry (a structure). The All row is a special row that applies to all structures in the Workspace. Each row has a set of buttons for doing operations on the structure. These buttons are actually menus: A (Action) perform various actions on the structure S (Show) show the structure or parts of the structure in different representations (line, stick, ball and stick, sphere, ribbon, cartoon) H (Hide) Hide various parts of the structure or its representations. L (Label) Label the structure or parts of it with various properties. C (Color) Color the structure or parts of it with various color schemes. The next few steps illustrate the use of these menus. 9. In the 2OT3 row, choose S Sticks. Bonds are shown in stick (tube) representation. 10. In the 2OT3 row, choose S As Lines. The sticks are no longer shown, and the bonds are represented by lines again. 11. In the 2OT3 row, choose H Waters. The water molecules are no longer shown in the Workspace. However, they are still counted as being in the Workspace, even though they are not visible. 12. In the 2OT3 row, choose S Lines. The water molecules are redisplayed. The representation applies to all atoms in the structure, and any hidden atoms are made visible again. 13. In the 2OT3 row, choose S As Ball and Stick. Atoms are now represented by balls, and bonds by sticks. 14. In the 2OT3 row, choose L Other Properties Stereochemistry. The chiral atoms are labeled R or S. Zoom in for a clearer view, then click Reset to zoom out again. 15. In the 2OT3 row, choose L Clear. The labels are removed. 16. In the 2OT3 row, choose C Color by Element Custom Color {C}HNOS. This color scheme colors all atoms by element, and applies a user-selected color to carbons. A color selector opens so you can choose a color for the carbons. BioLuminate 1.9 Quick Start Guide 5

12 Chapter 2: Using the Toggle Table 17. Click the gray cell in the lower right. The name of the color is shown in a tool tip. The structure is colored by element with gray carbons, blue nitrogens, red oxygens, orange sulfurs. For coloring, bonds are split in two, and colored by the color of the nearest atom. 18. In the 2OT3 row, choose C Custom Color All Atoms. 19. Click the gray cell in the lower right of the color selector. All atoms are colored gray. 20. In the 2OT3 row, choose C Color by Spectrum B Factors (Calpha). The carbon atoms are colored according to the PDB B factors (temperature factors). You can experiment further with the representation and color settings. There are also customizations of color and representation that can be applied to particular kinds of structures, such as protein interfaces. This structure has two chains, and hence an interface that can be displayed. (You can find the number of chains by looking in the status bar, below the Workspace, for the text Chn:2. The status bar gives information about the Workspace structure by default.) 21. In the 2OT3 row, choose A Preset Protein Interface. A progress bar is displayed briefly, and then the protein is redisplayed. The atoms in the interface region are displayed in ball and stick, while the rest of the protein is displayed in cartoon. Each chain is displayed in a separate color. So far the operations have been mostly on the entire structure. To perform operations on part of a structure, you have to select the atoms you want to work on. You can do this by picking atoms in the Workspace, either individually or as part of a group such as a residue, a molecule, or a chain. 22. Choose Edit Pick Mode Chains on the main menu bar. Now, when you pick (click on) atoms in the Workspace, the entire chain that the atom is part of is selected. The cursor in the Workspace changes to a box with a C next to it, to indicate that you are picking chains. 23. Click on one of the chains in the Workspace. You can click on the cartoon or on one of the atoms. The atoms are marked with yellow boxes, and a new row is added to the Toggle Table, labeled (Selection). This is the row that you can use to perform actions on the selected atoms. 6 Schrödinger Software Release

13 Chapter 2: Using the Toggle Table Figure 2.2. The BioLuminate main window with the selected chain. 24. In the (Selection) row, choose S As Lines. The chain that you selected is now shown with bonds as lines and atoms as points. 25. In the (Selection) row, choose A Copy to New Project Entry. The chain is copied to a new entry in the project, and a new row is added to the Toggle Table, labeled Selection001. This name is chosen as a unique name, but it doesn t provide any information about the structure. You can rename it. 26. In the Selection001 row, choose A Rename. The row name is replaced by a text box. 27. Click in the text box and type 2OT3 chain B. If the chain you selected is chain A rather than chain B, name it 2OT3 chain A. You can check which chain you selected by pausing the cursor over an atom in the Workspace. The identity of the atom is shown in the status bar below the Workspace, and includes the chain name, residue name and number, and atom name. BioLuminate 1.9 Quick Start Guide 7

14 Chapter 2: Using the Toggle Table 28. In the (Selection) row, choose A Modify Invert Within Any. The selection is inverted: all the atoms that were selected are now not selected, and the atoms that were not selected are selected. In other words, the other chain is now selected. 29. In the (Selection) row, choose A Copy to New Project Entry. The chain is copied to a new entry in the project, and a new row is added to the Toggle Table, labeled Selection002. If you wanted to remove the chain from the original protein rather than copying it, you could choose Extract to New Project Entry instead. 30. In the Selection002 row, choose A Rename. The row name is replaced by a text box. 31. Click in the text box and type 2OT3 chain A. Use B instead of A for the chain name if appropriate. You have now extracted the two chains from this protein into separate project entries. Each of these is included in the Workspace, as well as the original protein. 32. In the 2OT3 row, choose A Remove from Workspace. The original protein is no longer in the Workspace, and its row is no longer in the Toggle Table. The (Selection) row has also gone, because the selected atoms were all in the original protein. 33. Click the title of the 2OT3 chain A row in the Toggle Table. The atoms of this entry are hidden. As previously, the atoms are still considered to be in the Workspace, even if they are not visible. 34. Click the title of the 2OT3 chain A row in the Toggle Table again. The atoms of this entry are displayed again. 35. Click Include in Workspace. The Include in Workspace dialog box opens, listing all the entries in the project by title and entry ID. Entries that are already included in the Workspace have (Included) at the beginning of the row. 8 Schrödinger Software Release

15 Chapter 2: Using the Toggle Table 36. Select the 2OT3 row and click OK. Figure 2.3. The Include in Workspace dialog box The entry is included in the Workspace and the Toggle Table again. The selection you made earlier is not restored, because selection applies only to the Workspace, and only while the selected atoms are in the Workspace, and the selection was removed when this entry was removed from the Workspace. 37. In the All row, choose A Remove Everything from Workspace. All of the atoms are now removed, and only the All row remains in the Toggle Table. The exercises are complete, and the final step is to clean up by removing all the structures and closing the project. The project you have been working in is a temporary project, called a scratch project. 38. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and removed, and a new scratch project is opened. If you want to save and close the project instead of discarding it, you can click Save in the Save Scratch Project dialog box, then name the project in the file selector that opens. The newly saved project is closed. You can open it again from the File menu when you want to use it. You can convert a scratch project to a named, saved project at any point by choosing File Save As. Project data is automatically saved when it is updated, so you don t need to keep saving a project to preserve your data. For further information on the functions of the BioLuminate interface, you can use the help facility, either from the Help menu or by clicking the Help buttons. BioLuminate 1.9 Quick Start Guide 9

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17 BioLuminate Quick Start Guide Chapter 3 Chapter 3: Examining Sequences This exercise demonstrates the use of the Multiple Sequence Viewer to align, compare, and examine the sequences for a small set of homologous proteins. The first stage of the exercise is to import a single chain from each of three proteins. 1. Choose File Get PDB. The Get PDB File dialog box opens. 2. Enter 3max in the PDB ID text box. 3. Enter B in the Chain name text box. Figure 3.1. The Get PDB dialog box When you specify a chain, only that chain is imported. If you leave the Chain name text box blank, the entire protein is imported. To import a chain with a blank name, use ' '. 4. Click Download. Chain B of the 3MAX structure is imported into the project and displayed in the Workspace. 5. Choose File Get PDB. 6. Enter 1t64 in the PDB ID text box and click Download. The Chain name text box still contains B. As chain B of 1T64 is the chain that we want, the text in this text box does not need to be cleared or modified. Chain B of the 1T64 structure is imported into the project and added to the Workspace. 7. Choose File Get PDB. BioLuminate 1.9 Quick Start Guide 11

18 Chapter 3: Examining Sequences 8. Enter 1c3r in the PDB ID text box and click Download. Chain B of the 1C3R structure is imported into the project and added to the Workspace. All three proteins are now in the Workspace. 9. Choose Tools Multiple Sequence Viewer. The Multiple Sequence Viewer panel opens, showing the sequences for the three proteins. The sequences are colored by residue type, and each sequence is annotated with its secondary structure assignment (labeled SSA on the left). Note that all the sequences start at the left side and have no gaps. Figure 3.2. The Multiple Sequence Viewer panel, initial view. The next step is to align the three sequences, with 3MAX as the query. 10. Right-click on 3MAX_B in the left pane and choose Set as Query Sequence. This sequence is placed at the top of the sequence list, and it is marked in blue to indicate that it is the selected sequence. It is also the query sequence, and its identity is reported in the status bar at the bottom of the panel, with information on the number of sequences. The shortcut menu has a number of actions that you can perform on the sequence. 12 Schrödinger Software Release

19 Chapter 3: Examining Sequences 11. Shift-click on the last sequence in the left pane. The three sequences are now selected. 12. Click the Multiple Alignment toolbar button. The alignment is performed on the selected sequences. A dialog box is displayed briefly as the alignment is done. When the alignment finishes, you can see that there are now gaps in the displayed sequences. It is a little easier to compare the sequences if the annotations are removed. 13. Choose Annotations Clear Annotations. The secondary structure assignment is removed. You can also hide annotations using the turner for the tree view in the left pane. 14. Move the pointer slowly over one of the gaps, so that you can see the tooltip information for each residue. The position in the sequence viewer is given first, followed by the 3-letter residue name and number. The residue numbers have not changed, only the position in the viewer. 15. Click the Color Matching Residues Only toolbar button. The coloring of the second and third sequences changes so that only the residues that match the query sequence are colored; the rest are given a white background. The query sequence remains colored. This tool makes it easy to identify the residues that match the query. 16. Click the Weight Colors by Alignment Quality toolbar button. The intensity of the coloring changes to reflect the fraction of matching residues at each residue position. Where all three sequences have the same residue, the color intensity is at its maximum; where only two residues match at a position, the color is medium, and where only one matches (in the query sequence) the color is light. This tool makes it easy to identify the positions that have the most matches. BioLuminate 1.9 Quick Start Guide 13

20 Chapter 3: Examining Sequences Figure 3.3. Coloring of matching residues. The Multiple Sequence Viewer has a number of useful annotations that can be displayed below the sequence. 17. Choose Annotations Ligand Contacts. The structure is analyzed to find the contacts of the ligand with the protein. After a while, the annotation is displayed. The ligand is represented by a row, with positions marked in red where there are close (< 4 Å) contacts between heavy atoms, and in orange for other contacts (< 6 Å). The rest of the row is gray. 18. Choose Annotations B Factor. The temperature factor is shown as a bar chart below the sequence. 14 Schrödinger Software Release

21 Chapter 3: Examining Sequences Figure 3.4. Ligand contact and B-factor annotations. The Multiple Sequence Viewer contains many more features, for manual alignment and editing of sequences, selection of residues, homology modeling, and prediction of structural features. For more information, see the online help or the Multiple Sequence Viewer document. A restricted version of the Multiple Sequence Viewer is used in many of the BioLuminate panels. 19. Close the Multiple Sequence Viewer panel. 20. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. BioLuminate 1.9 Quick Start Guide 15

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23 BioLuminate Quick Start Guide Chapter 4 Chapter 4: Identifying Reactive Residues This exercise demonstrates how to identify reactive residues in a protein, which is imported from the PDB. The identification is done by matching residue patterns in the sequence. 1. Choose Tools Protein Preparation. The Protein Preparation Wizard panel opens. This panel is available from both the Tools menu and the Tasks menu. Figure 4.1. The Protein Preparation Wizard panel. 2. Enter 2pcy in the PDB text box, and click Import. The 2PCY structure is imported into the project and displayed in the Workspace. This panel provides a wide range of options for preparing proteins for modeling: fixing structural defects and making assignments required by modeling applications. For this exercise, the default settings will be used. BioLuminate 1.9 Quick Start Guide 17

24 Chapter 4: Identifying Reactive Residues 3. Click Preprocess. The structure is processed, and a new structure is added to the project and displayed in the Workspace, replacing the imported structure. You can close the Protein Preparation Wizard panel. 4. Choose Tools Reactive Residue Identification. The Reactive Protein Residues panel opens. The predefined reaction types are selected by default. You can define your own (click Edit), but for this exercise the defaults will be used. 5. Click Analyze Workspace. Figure 4.2. The Reactive Protein Residues panel. The structure in the Workspace is analyzed to identify residues that match the patterns defined for deamidation, oxidation, glycosylation, and proteolysis. The results are listed in the table, and the sites are marked with spheres in the Workspace. The spheres are colored according to the reaction type, and the color legend for the spheres is given below the table. Also below the table are some tools for filtering the residues shown in the table and in the Workspace. 18 Schrödinger Software Release

25 Chapter 4: Identifying Reactive Residues Figure 4.3. Reactive residue sites in 2PCY. 6. Enter 80 in the Solvent exposure >= text box. The list is reduced to two proteolysis sites, which have a high solvent exposure. 7. Click the row with 90% exposure. The view zooms in to this residue, whose side chain has little contact with the rest of the protein. 8. Set the solvent exposure threshold back to Choose Oxidation from the Show option menu. Only one residue is listed in the table. 10. Choose All from the Show option menu. 11. Click on the heading of the Exposure column. The residues are sorted by exposure. The oxidation site is the one that has the lowest solvent exposure. 12. Close the Reactive Protein Residues panel. 13. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. BioLuminate 1.9 Quick Start Guide 19

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27 BioLuminate Quick Start Guide Chapter 5 Chapter 5: Identifying Consensus Waters This exercise demonstrates the use of the Consensus Viewer to identify conserved non-protein structural elements in a set of homologous proteins in this case, waters. 1. Choose Tools Protein Preparation. The Protein Preparation Wizard panel opens. 2. Enter 2pcy in the PDB text box, and click Import. The 2PCY structure is imported into the project and displayed in the Workspace. 3. Deselect Delete waters beyond N Å from het groups. This exercise uses the waters, so they should not be deleted. Figure 5.1. The Protein Preparation Wizard panel. BioLuminate 1.9 Quick Start Guide 21

28 Chapter 5: Identifying Consensus Waters 4. Click Preprocess. The structure is processed, and a new structure is added to the project and displayed in the Workspace, replacing the imported structure. You can close the Protein Preparation Wizard panel. 5. Choose Tools Protein Consensus Viewer. The Consensus Visualization panel opens. 6. Click Import and choose From Workspace. The structure in the Workspace is imported into the sequence viewer in the panel. Figure 5.2. The Consensus Visualization panel after import. 7. Click Find and Align Homologs. The Blast Search Settings dialog box opens. 8. From the Database option menu, choose NCBI PDB (non-redundant). 9. Choose a BLAST Server option. 22 Schrödinger Software Release

29 Chapter 5: Identifying Consensus Waters If you have a local installation of the BLAST database, you can choose Local. Otherwise, choose Remote (NCBI). 10. Click Start Job. A Job Progress dialog box replaces the Blast Search Settings dialog box, and displays the log file from the BLAST search. After a few minutes, the job finishes and the BLAST Search Results dialog box opens, with the results of the search. The top ten results are selected in the table. 11. Click Incorporate Selected Rows. Figure 5.3. The BLAST Search Results dialog box. If you do not have a local installation of the BLAST or PDB databases, the search is done on the web, and a warning is displayed: Multiple Sequence Viewer is attempting to access a remote server. Would you like to continue? You can select Do not ask this question again, to prevent it from opening each time a structure is downloaded, and click OK. If an information box opens stating that problems were found when importing a structure, you can select Do not show this dialog again to prevent it from opening for each structure that has problems, and click OK. BioLuminate 1.9 Quick Start Guide 23

30 Chapter 5: Identifying Consensus Waters The ten homologs are added to the sequence viewer and displayed in the Workspace. All atoms are selected in all of the homologs. 12. In the Water section, choose Consensus from the Display option menu. The consensus water structures are displayed with the proteins in the Workspace, represented as spheres and outlined. In this case there is only one consensus water structure, because consensus is defined as having a water at this position in at least 60% of structures. This percentage is specified under Define consensus. Figure 5.4. The Consensus Visualization panel with results. 13. Change the percentage under Define consensus to 50. More consensus water structures are shown, at several locations. At some locations, the spread in the positions of the waters is greater than others. Some spheres have hydrogens in white, others do not. This is because hydrogen atoms are present on the waters in the 2PCY structure, but are not present in the other structures. 14. In the All row of the Toggle Table, choose A Remove Everything from Workspace. 24 Schrödinger Software Release

31 Chapter 5: Identifying Consensus Waters 15. In the Consensus Visualization panel, click the check box for 2PCY_A in the sequence viewer. The 2PCY protein is displayed, with its consensus waters. The color scheme (red oxygen, white hydrogen) was set by the protein preparation process, and is not altered by this panel. 16. Click the check box for 1JXG_A in the sequence viewer. The 1JXG protein is displayed with its consensus waters, along with 2PCY. The waters for 1JXG are represented by red spheres for the oxygen atoms. There are no hydrogens on the 1JXG waters. The spheres overlap with the waters for 2PCY at four of the five water locations. 17. Click the check box for 2PCY_A again in the sequence viewer. The 2PCY protein is removed from the Workspace, and only the 1JXG protein remains, with its consensus waters shown as red spheres. The identity of any consensus water can be ascertained by moving the cursor over the consensus water sphere and viewing the text in the status bar, below the Workspace. 18. Close the Consensus Visualization panel. 19. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. BioLuminate 1.9 Quick Start Guide 25

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33 BioLuminate Quick Start Guide Chapter 6 Chapter 6: Homology Modeling of Proteins This exercise demonstrates how to run a simple homology model of a protein. A structure from the PDB is used, and a template is chosen that represents a realistic model for an unknown protein. If you are interested in homology modeling of antibodies, see Chapter If the Workspace is not empty, in the Toggle Table panel click the A button in the All row and choose Remove Everything from Workspace. 2. Choose File Get PDB. 3. Enter 2pcy in the PDB ID text box, and click Download. The 2PCY structure is displayed in the Workspace. 4. Choose Tasks Homology Modeling Simple Homology Modeling in the main window. The Homology Model panel opens. Figure 6.1. The Homology Model panel, initial view. 5. Click the Query button and choose From Workspace. The box to the right of the button displays the text ( Query structure ) and the title of the Workspace structure. BioLuminate 1.9 Quick Start Guide 27

34 Chapter 6: Homology Modeling of Proteins 6. Click the Template button and choose BLAST Homology Search. The Blast Search Settings dialog box opens. This dialog box allows you to change parameters of the BLAST search. For this exercise (and for most purposes), the defaults can be used. 7. Click Start Job. A Job Progress dialog box replaces the Blast Search Settings dialog box, and displays the log file from the BLAST search. After a few minutes, the job finishes and the BLAST Search Results dialog box opens, with the results of the search. The best homolog is selected. For this exercise, we will choose one of the lower-ranking homologs to illustrate what would happen in a real situation with an unknown structure. Figure 6.2. The BLAST Search Results dialog box. 8. Scroll down to 1M9W_Aand select it. This homolog has an identity and a similarity that are more typical for an unknown structure. 28 Schrödinger Software Release

35 Chapter 6: Homology Modeling of Proteins 9. Click Select Template. If you do not have a local installation of the BLAST or PDB databases, a warning is displayed by default: Multiple Sequence Viewer is attempting to access a remote server. Would you like to continue? If you have not already turned this warning off, Select Do not ask this question again, to prevent it from being displayed each time a structure is downloaded, and click OK. The template is selected and the BLAST Search Results dialog box closes. The table rows in the Homology Model panel are filled in with information on the template. Note the rankings: the similarity and homology are considered good, but the identity is only fair. 10. Click Run. Figure 6.3. The Homology Model panel after selecting the template. The job includes alignment of the template and the query using ClustalW, and the structure is built on the basis of the template and an analysis of structural elements in the PDB for non-templated regions (a knowledge-based selection of the coordinates). After a few minutes, the job finishes. The model is added to the Workspace. The cartoon is colored by how the template was used: dark blue for residues for which all coordinates were taken from the template, cyan for residues for which the backbone was taken from the template, and red for residues that were entirely modeled, not using the template. The buttons in the Homology results section of the Homology Model panel are now available. BioLuminate 1.9 Quick Start Guide 29

36 Chapter 6: Homology Modeling of Proteins 11. Click Examine Model Quality. The Protein Structure Quality Viewer panel opens, with the Ramachandran Plot tab displayed. The model is the only structure in the Workspace, and the residues are colored by the plot region that they are in. 12. Pause the pointer over a point in the disallowed region. The identity of the residue and its angles are displayed at the top right of the plot, and the residue is selected and highlighted in the Workspace. If you move the pointer off the point, the residue is no longer highlighted, and the Selection row in the Toggle Table is dimmed, indicating that it is no longer selected. Selected point Figure 6.4. Protein Structure Quality viewer with point selected in Ramachandran Plot, and Workspace showing selected residue. 30 Schrödinger Software Release

37 Chapter 6: Homology Modeling of Proteins 13. Now click on the same point. The view zooms in to show the residue centered in the Workspace with its nearest neighbors. This happens when you click on a point but not when you pause over a point. The selection in the Selection row is now persistent: it remains until you make a new selection. If you pause over a different point, its details are shown to the top right of the plot and it is highlighted and selected in the Workspace (although it may be off screen), but when you move the pointer off the new point, the details of the point you clicked on are displayed again and it is highlighted and selected again. 14. In the Protein Report tab, choose Backbone Dihedrals from the Display option menu. The table is populated with values of the backbone dihedrals and the derived G-factor, which measures the likelihood of the combination of dihedrals, and the G-factor is plotted below the table for all the residues in the protein. Figure 6.6. Protein Structure Quality viewer with protein report for backbone dihedrals, and Workspace showing residues selected in dihedral plot. BioLuminate 1.9 Quick Start Guide 31

38 Chapter 6: Homology Modeling of Proteins 15. Move the pointer across the plot, and pause over one of the large negative peaks. The identity of the residue for each plot point is displayed to the top right of the plot with its G-factor. For the large negative peaks, the text disallowed is displayed instead of the G-factor. 16. Drag the dotted blue vertical lines of the plot inward so that they are on either side of the large peak belo row number Drag the top dotted red line down so that it is just below the 9 mark on the vertical axis. As you drag, the residues in the area between the blue and red lines are selected, and the Workspace view zooms to these residues. On the plot, the background of the area outside the rectangle bounded by these lines is colored gray. This is a way of selecting residues that have particular values of a property. 18. Close the Protein Structure Quality Viewer panel. 19. In the Selection row of the Toggle Table choose A Delete Selection. The selection row disappears, indicating that there is now no selection of atoms in the Workspace. To further check the accuracy of the model, it can be visually compared with the X-ray structure in the Workspace. The homology model should be the only structure in the Workspace. It is titled Model of 2PCY built on 1M9W. 20. Click Include in Workspace. The Include in Workspace panel opens, listing the titles and IDs of the project entries that you can include. 21. Choose the 2PCY entry and click OK. The X-ray structure is now in the Workspace. 22. In the All row of the Toggle Table choose C Auto All Atoms by Object. 23. In the All row of the Toggle Table choose S As Cartoon. 24. On the main menu bar, choose Tools Protein Structure Alignment. The Protein Structure Alignment panel opens over the Toggle Table. The default settings should be appropriate for this task: Use proteins from is set to Workspace (included entries). The text box in the Reference residues section contains all. Use same ASL as reference residues is selected under Residues to align. 32 Schrödinger Software Release

39 Chapter 6: Homology Modeling of Proteins 25. Click Align. After a short while, the proteins are aligned. The alignment score and the RMSD are reported in the Protein Structure Alignment Results panel. The Workspace should look something like Figure 6.7. If the structures are not visible, click the Zoom button in the Toggle Table. Figure 6.7. The model and the X-ray structure of 2PCY. 26. Close the Protein Structure Alignment panel and the Protein Structure Alignment Results panel. 27. Rotate the structure around to compare the X-ray structure (green in Figure 6.7) with the model (cyan). If the results are not on screen, click Zoom. Overall agreement between the predicted model and the X-ray structure is generally very good. Differences in the secondary structural elements between the two are mostly small, with the exception of the loop formed by residues This is not unexpected, given the lack of template residues (a sequence alignment gap) between residues 47 and 50. An improved model for this loop could be set up and run in the Refinement panel by clicking Refine Loops in the Homology Model panel. Loop prediction can take a long time, so it is not included as part of this exercise. BioLuminate 1.9 Quick Start Guide 33

40 Chapter 6: Homology Modeling of Proteins 28. Close the Homology Model panel. 29. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. 34 Schrödinger Software Release

41 BioLuminate Quick Start Guide Chapter 7 Chapter 7: Scanning for Residue Mutations This exercise demonstrates how to scan a protein for potential residue mutations and compare the relative binding affinity of two protein chains for each of the mutants. The protein must first be prepared, as only two of the chains will be kept for this exercise. If the Workspace is not empty, in the Toggle Table panel click the A button in the All row and choose Remove Everything from Workspace. 7.1 Preparing the Protein 1. Choose Tools Protein Preparation. The Protein Preparation Wizard panel opens. 1. Click this button. 3. Click this button. 2. Select chains. Figure 7.1. Deleting chains. BioLuminate 1.9 Quick Start Guide 35

42 Chapter 7: Scanning for Residue Mutations 2. Enter 1brs in the PDB text box, and click Import. The 1BRS structure is imported into the project and displayed in the Workspace. For this exercise, the unwanted chains will be removed first, then the structure will be processed. 3. In the Review and Modify tab, click Analyze Workspace. The tables of chains, waters, and het groups are filled in. This is done automatically when you process the structure in the Import and Process tab. 4. Select chains B, C, E, and F in the table of chains. 5. Click Delete. The selected chains are deleted, leaving just chains A and D. Now the trimmed structure can be processed. 1. Select this option. 1. Click this button. Figure 7.2. Preprocessing the protein with side-chain addition. 6. In the Import and Process tab, select Fill in missing side chains using Prime. This protein has some side-chain coordinates missing in the PDB structure. Adding them at this stage performs a quick addition of the side chains. Refinement of their locations will be done later. 36 Schrödinger Software Release

43 Chapter 7: Scanning for Residue Mutations 7. Click Preprocess, and confirm the addition of the side chains if prompted. The structure is processed, and a new structure is added to the project and displayed in the Workspace, replacing the imported structure. The Protein Preparation - Problems dialog box opens, reporting atom clashes. This problem will be fixed later in the process. 8. Click OK to dismiss the Protein Preparation - Problems dialog box. 9. In the Refine tab, click Optimize in the H-bond assignment section. Optimizing the hydrogen bonding is important because X-ray structures do not usually have enough resolution to fix the orientation of terminal amides or histidines, or the orientation of hydroxyls and thiols. When the optimization finishes, a new structure is added to the project. The structure is labeled with the changes that were made to terminal amides and histidines, to make it easy to examine these changes if you wish. 1. Click this button. 2. Click this button. Figure 7.3. Refining the protein structure. BioLuminate 1.9 Quick Start Guide 37

44 Chapter 7: Scanning for Residue Mutations 10. In the Protein Preparation Wizard panel, click Minimize. A restrained minimization is done, which removes the atom clash and relaxes the side chains and other modifications made to the protein. The results are added as a new entry to the project. You can click View Problems to verify that the clash has gone. The protein is now ready for use. 11. In the 1BRS row of the Toggle Table, choose L Clear. The labels are removed. 7.2 Setting Up the Mutations 1. Choose Tasks Residue Scanning Perform Calculations in the main window. The Residue Scanning panel opens. 1. Click this button. 2. Select residues. 3. Click this button. Figure 7.4. Setting up the residue scanning job. 38 Schrödinger Software Release

45 Chapter 7: Scanning for Residue Mutations 2. Check that Import structure from is set to Workspace, and click Import. The structure in the Workspace is analyzed and the residues table is filled in with all the residues in the protein. 3. Select the rows for the following residues: A:27 (LYS), A:54 (ASP), A:58 (ASN), A:59 (ARG), A:60 (GLU), A:73 (GLU), A:87 (ARG), A:102 (HIE). 4. Check that Mutate is set to Selected residues and that the residue in the to option menu is set to ALA. 5. Click Apply. The mutation is set to alanine in the marked rows. The text message at the bottom of the tab should read Will mutate 8 of 195 residues; Number of mutations: Setting Options and Running the Job 1. Click the Options tab. 2. From the Refinement option menu, choose Side-chain prediction with backbone sampling. This option allows for some movement of the backbone in response to the change in side chain. The rest of the settings can be left as they are. 3. Click the Settings button to open the Job Settings dialog box. 4. Choose a host from the Host option menu. If you chose a multiprocessor host, you can distribute the job over multiple processors. For this job, you can specify a maximum of 8 subjobs, because there are 8 mutations, and you can also specify up to 8 processors. 5. Click Run to run the job. A status bar showing the progress of the job is displayed above the Job toolbar. The job takes about an hour to run on a single processor. 7.4 Examining the Results When the job finishes, the Residue Scanning Viewer panel opens, displaying changes in properties for each mutation, and a graph of one of the properties. BioLuminate 1.9 Quick Start Guide 39

46 Chapter 7: Scanning for Residue Mutations Figure 7.5. The Residue Scanning Viewer panel. 1. Choose Δ Affinity from the Graph property option menu. The graph shows the change in affinity of the mutated chain for the other chain on mutation, as a function of the row number in the table. Note that some mutations improve the affinity between the chains, while others destabilize the complex. 2. Close the Residue Scanning panel and the Residue Scanning Viewer panel. 3. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. 40 Schrödinger Software Release

47 BioLuminate Quick Start Guide Chapter 8 Chapter 8: Locating Possible Mutations for Disulfide Bridges This exercise demonstrates how to run a cysteine mutation calculation to locate and rank possible disulfide bridges. A structure from the PDB that already has a disulfide bridge is used. After preparation of the protein, the cysteine residues are mutated to alanine, and the Cysteine Mutation panel is then used to locate and restore the bridge. 1. If the Workspace is not empty, in the Toggle Table panel click the A button in the All row and choose Remove Everything from Workspace. 2. Choose Tools Protein Preparation. 3. Enter 1vhu in the PDB text box, and click Import. The 1VHU structure is imported into the project and displayed in the Workspace. This structure is missing a side chain, which can be filled in. 4. Select Fill in missing side chains using Prime. 5. Click Preprocess, and confirm the addition of the side chains when prompted. The structure is processed, and a new structure is added to the project and displayed in the Workspace, replacing the imported structure. The Problems dialog box opens at the Alternate Positions tab, because there are alternate positions for some residues. 6. Select the first row in the table and click Commit until the table is empty. 7. Click OK. 8. In the Refine tab, click Optimize in the H-bond assignment section. Optimizing the hydrogen bonding is important because X-ray structures do not usually have enough resolution to fix the orientation of terminal amides or histidines, or the orientation of hydroxyls and thiols. When the optimization finishes, a new structure is added to the project. The structure is labeled with the changes that were made to terminal amides and histidines, to make it easy to examine these changes if you wish. 9. In the 1VHU row of the Toggle Table, click L and choose Clear. The labels are removed. 10. Choose Tasks Residue and Loop Mutation. BioLuminate 1.9 Quick Start Guide 41

48 Chapter 8: Locating Possible Mutations for Disulfide Bridges Figure 8.1. The Residue and Loop Mutation panel. 11. Scroll the sequence viewer until you can see the first cysteine of the disulfide bridge, and select this cysteine. The disulfide bridge is marked with a black line connecting the two cysteines. When you click on it, the column is marked in blue, extending across the annotations as well as the sequence. The residue is also selected in the Workspace. 12. Click Workspace Selection in the Original from section. The sequence selection is cleared, and the details of the structure and the mutation are displayed lower in the panel. The selection in the Mutated to section is ALA, which is the residue we want to mutate to, so no further changes are needed. 13. Click Run. After a short while, the mutated structure replaces the Workspace, with the alanine drawn as sticks. 42 Schrödinger Software Release

49 Chapter 8: Locating Possible Mutations for Disulfide Bridges 14. Choose Reset Panel from the Settings (gear) button menu. The previous structure is cleared, and the sequence is replaced by the sequence of the structure that is currently in the Workspace, which is the newly created mutant. Note that the annotation for the disulfide bridge has gone, because one of the residues has been mutated. 15. Scroll the sequence viewer to find the next cysteine, CYS154, and select it. 16. Click Workspace Selection. 17. Click Run. 18. Close the Residue and Loop Mutation panel. 19. In the Protein Preparation Wizard panel, click Minimize in the Refine tab. When the job finishes, you can close the Protein Preparation Wizard panel. The mutated protein is ready for the rest of the exercise. 20. Choose Tasks Cysteine Mutation. The Cysteine Mutation panel opens. 21. Click Analyze Workspace. The structure in the Workspace is analyzed to identify residues that could be mutated to form or to break disulfide bonds. Of the 98 pairs identified, only one pair involves a single cysteine, and the rest involve two non-cysteine residues, including the pair you mutated. Because the protein has no disulfide bridges, there are no candidates for removing the bridge by mutation, but the analysis identifies such Cys-Cys pairs if they are present. 22. From the X->Cys replacement residues menu, choose Conservative. The text box shows five residues, GLY, ILE, LEU, VAL, ALA, and the number of residues in the table is reduced from 98 to In the Beta carbon distance cutoff text box, type 4.0. The number of pairs is now 6. (You might see a different number, as one of the pairs has a distance that is very close to the cutoff.) The restrictions on the residues and the distances were added to make the run time smaller. Running all 98 mutations found with the default conditions would take about 3 hours. 24. Place the pointer in the table and press CTRL+A ( A). All of the remaining 6 table rows are selected, and the selection is noted below the table. The residues are highlighted in the Workspace structure in ball-and-stick representation. BioLuminate 1.9 Quick Start Guide 43

50 Chapter 8: Locating Possible Mutations for Disulfide Bridges 25. Under Minimization shell, select None. Figure 8.2. The Run tab of the Cysteine Mutation panel. 26. From the Refinement option menu, choose Implicit solvent minimization. 27. Click Run to start the job. There is no need to change the default job settings. The job takes about 15 minutes. Its progress is displayed in a progress bar above the Job toolbar. When the job finishes, the results are displayed in the Results tab. 44 Schrödinger Software Release

51 Chapter 8: Locating Possible Mutations for Disulfide Bridges Figure 8.3. The Results tab of the Cysteine Mutation panel. 28. Scroll the table to the right and click on the heading of the Weighted Score column. The residue pairs are sorted by this score. Four of the mutations have scores of around 400, while the other two have scores around The large scores have an offset added because they fail to meet one of the cutoff criteria on the change in interaction energy (< 5.0) or strain energy (< 35.0). 29. Scroll back to the left. Note that the original cysteine pair (A:111-A-154) is ranked at the top. BioLuminate 1.9 Quick Start Guide 45

52 Chapter 8: Locating Possible Mutations for Disulfide Bridges 30. Click the A:111-A:154 row. The residue pair is displayed in the Workspace, colored by element and represented in ball-and-stick. The original protein is also present, colored gray. All of the mutated structures are included in the project. The mutation is identified by the res pair property. You can view the properties in the Project Table, which you open by pressing CTRL+T ( T) in the main window. 31. Close the Cysteine Mutation panel. 32. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. 46 Schrödinger Software Release

53 BioLuminate Quick Start Guide Chapter 9 Chapter 9: Modeling an Antibody This exercise demonstrates how to model an antibody by homology modeling, using a database of antibody structures to locate homologs, and then humanize the antibody. It uses a known antibody structure, 1fsk, which is removed from the antibody database so that it can be treated as an unknown antibody, and the model can be compared with the crystal structure. 9.1 Importing the Structure 1. Choose Tasks Antibody Modeling Prediction in the main window. The Antibody Prediction panel opens. On the left is a labeled diagram of an antibody; on the right are three tabs for setting up and performing the modeling. 2. Click the heavy variable region of the diagram and choose From PDB ID from the menu. This region is marked when you pause the pointer over it. When you click, a menu is displayed. When you choose the menu item, the Enter PDB ID dialog box opens. 3. Type 1fsk in the text box and click OK. The protein is imported and analyzed. The PDB structure is a dimer. After a while, the Choose Heavy Region dialog box opens, prompting you to choose the chain to use. 4. Choose chain C from the menu and click OK. The heavy variable region is colored green to indicate that it has been assigned. 5. Click the light variable region of the diagram and choose From Selected Entries in the Project Table. The PDB sequence and structure has already been imported, so you can use it for the light chain. It is selected in the Project Table by default. The protein is analyzed for light chains. After a while, the Choose Light_kappa Region dialog box opens, prompting you to choose the chain to use. 6. Select chain B and click OK. The light variable region is colored blue to indicate that it has been assigned, and the text prompting you to import the regions is removed see Figure 9.1. Figure 9.1. (next page) The Antibody Prediction panel. Top: Diagram showing chain label. Center: Import menu. Bottom: After importing both chains. BioLuminate 1.9 Quick Start Guide 47

54 Chapter 9: Modeling an Antibody 48 Schrödinger Software Release

55 Chapter 9: Modeling an Antibody 9.2 Filtering the Templates To Be Used The next task is usually to select the database to use in the search for homologs for the framework region. In this tutorial, the default database from the installation is used, so no action is needed to select a database. However, the antibody you imported must be removed from the database search, so the modeling of an unknown antibody can be simulated. 1. Click Filter Search Results. The Filter Search Results dialog box opens. It allows you to restrict the search by filtering the database on the values of one or more properties. The property you will use for this exercise is the PDB ID. Figure 9.2. The Filter Search Results dialog box. 2. In the Property text box, type p, then choose PDB ID from the completion list that is displayed. 3. From the option menu next to it, choose Not =. 4. In the next text box, type 1fsk. It does not matter whether you use upper case or lower case for the PDB ID. 5. Click Add. The Filtering definitions and criteria area displays the criterion you just defined, and also reports the number of matches to the filter, 1582, which is one fewer than the 1583 structures in the full antibody database. BioLuminate 1.9 Quick Start Guide 49

56 Chapter 9: Modeling an Antibody 6. Click OK. 9.3 Searching for Templates The next step is to search for a template for the framework region. 1. Click Search. When the search finishes, the table is filled in with the results. 1fsk is not in the list, because it was removed by the filter. The highest-scoring result is 1i3g, which is selected by default, and will be used for this exercise. Figure 9.3. The Antibody Prediction panel after searching for templates. 2. Click Accept. The template for the selected row in the results table is accepted as the template, and is imported into the project. After a short while, the Basic Loop Model tab is displayed. 9.4 Generating the Basic Loop Models In the Basic Loop Model tab you can choose whether to model the loops using the antibody database or use the input coordinates for the loops. If you model the loops, you can select a template to use for each loop that you model by double-clicking the Loop Template table cell, and choosing a cluster in the Loop Clusters panel. By default the largest cluster of loops of the appropriate length is chosen automatically, and the loop from this cluster that is most similar to the query is used. In this exercise you will use the defaults, which includes generating a single model. 50 Schrödinger Software Release

57 Chapter 9: Modeling an Antibody Figure 9.4. The Antibody Prediction panel, Basic Loop Model tab. 1. Click Generate Loop Models. After a few minutes, the models are generated, and the Model to view menu is populated. This model can be used as it is, but in real situations, you might want a more accurate prediction of the H3 loop, which you can set up and run in the Advanced Loop Model tab. 9.5 Preparing the X-Ray Structure for Comparison To check the accuracy of the homology model, you will compare this structure with the X-ray structure. To do this, the X-ray structure needs to be pruned down to the size of the modeled structure. This will be done using the Workspace sequence viewer. 1. If the Workspace is not empty, in the All row of the Toggle Table panel choose A Remove Everything from Workspace. 2. Click Include in Workspace. The Include in Workspace panel opens, listing the titles and IDs of the project entries that you can include. 3. Choose the first 1FSK entry and click OK. The X-ray structure is now in the Workspace. 4. If the sequence viewer is not already displayed, choose Edit Settings Show Sequence Viewer on the main menu bar. 5. In the sequence viewer, click the first residue in chain B, then scroll down and shift-click the last residue in chain B. BioLuminate 1.9 Quick Start Guide 51

58 Chapter 9: Modeling an Antibody Chain B is now selected. Yellow markers are displayed in the Workspace on the chain and the chain is highlighted in the sequence viewer. 6. Control-click the last residue in chain C, then scroll up and shift-control-click the first residue in chain C. (Use command-click and shift-command-click on a Mac.) Both chains B and C should now be selected. 7. Right-click on the selection in the sequence viewer and choose Invert Selection. The selection now includes all chains but B and C. 8. Press the DELETE key. (Small keyboard Mac: FN+DELETE.) The selected chains are deleted and only chains B and C remain. These are the chains that were modeled, but they are longer than the chains in the model. 9. In the Toggle Table panel, click Include in Workspace. 10. Choose the Antibody prediction (model 1) entry and click OK. Both structures are now in the Workspace. 11. In the sequence viewer, scroll to the last residue in chain L of the model. Chain L has the same sequence as chain B in the X-ray structure up to this point. Chain B has more residues, which will be deleted. 12. Click the next residue in chain B. 13. Scroll to the end of chain B and shift-click the last residue. 14. Press the DELETE key. 15. Scroll to the last residue in chain H of the model. Chain H has the same sequence as chain C in the X-ray structure, but the numbering is slightly different because chain H comes from the homology model, and uses the numbering of the template, with insertion codes for any insertions. 16. Click the next residue in chain C. 17. Scroll to the end of chain C and shift-click the last residue. 18. Press the DELETE key. The X-ray structure is now pruned down to the same size as the model. 52 Schrödinger Software Release

59 Chapter 9: Modeling an Antibody 9.6 Comparing the Model with the X-Ray Structure You can now perform a structural alignment, to see how much the model structure differs from the X-ray structure. To facilitate the comparison, some changes must be made to the display. 1. In the All row of the Toggle Table, choose C Auto All Atoms by Object. 2. In the All row, choose S As Cartoon. Now the alignment can be done: 3. On the main menu bar, choose Tools Protein Structure Alignment. The Protein Structure Alignment panel opens in a new tab over the Toggle Table. The default settings should be appropriate for this task: Use proteins from is set to Workspace (included entries). The text box in the Reference residues section contains all. Use same ASL as reference residues is selected under Residues to align. 4. Click Align. After a short while, the proteins are aligned. The alignment score and the RMSD are reported in a separate panel, Protein Structure Alignment Results, and both indicate that the alignment is good. The Workspace should look something like Figure 9.5. Note the patchy appearance of the ribbons: this is because they are almost in the same location, indicating that the model is very good. Likewise, the loops of the model follow the loops of the X-ray structure closely. 5. Close the Protein Structure Alignment panel and the Antibody Prediction panel. BioLuminate 1.9 Quick Start Guide 53

60 Chapter 9: Modeling an Antibody Figure 9.5. Results of the comparison. 54 Schrödinger Software Release

61 Chapter 9: Modeling an Antibody 9.7 Humanizing the Antibody The final exercise is to humanize the antibody, which involves selection of residues to mutate. The residues can be selected on the basis of homology or structural criteria. If you do not want to continue, you can close the project and discard it. Otherwise, continue with the following steps. 1. In the 1FSK row of the Toggle Table, choose A Remove from Workspace. The truncated X-ray structure is removed, and the antibody model remains. 2. Choose Tasks Antibody Modeling Humanization Residue Mutation. The Antibody Humanization: Residue Mutation panel opens, with the Residues tab displayed. Most of the controls are inactive. 3. Click Import. After a short delay, the residue table in the Residues tab is filled in. If you want to limit the analysis to a part of the antibody, you can select the relevant part in the Workspace first, and then choose Workspace (selected residues only) before clicking Import. 4. Click Homology Criteria. The Homology Suggestions dialog box opens, with the heavy chain displayed in the sequence viewer. Here you look for homologs that will provide a set of mutations for humanizing the antibody. 5. Click Search Antibody Database for Homologs. When the search is done, the sequence viewer includes the results of the search. Next, the homologs must be aligned so that selection of residues can be done on the basis of related residue positions. 6. Click Align Homologs. The homologs are aligned. You may see gaps in the sequences in the sequence viewer. These are added as part of the alignment, but they do not affect the original sequences or the structures. The lower half of the tab contains tools for selecting the regions that are searched for possible mutations and criteria for selecting residues by variability or conservation among the homologs, or by solvent accessibility or contact with other residues. In this exercise, the default regions (Non-CDR) and selection criteria will be used. BioLuminate 1.9 Quick Start Guide 55

62 Chapter 9: Modeling an Antibody Figure 9.6. The Homology Suggestions dialog box. 7. Select Solvent accessible surface area in the Parent structure 3D criteria section. 8. Check that the settings for this option are > 70%. 9. Click Save. The residues that match all the criteria are selected for mutation, and a message is displayed indicating how many residues were selected. 10. In the Residues tab, scroll through the table to locate the selected residues. Nine residues are selected, three in the heavy chain and six in the light chain. For each selected residue, mutations are specified based on the variability in the homologous proteins. The total number of mutations is reported lower in the panel. You can change them by clicking in the table cell, and selecting the residues from the menu that is displayed in the cell. Click again in the table (e.g. on the row number) to dismiss the menu. The mutations for a residue will be cleared in the next step. 56 Schrödinger Software Release

63 Chapter 9: Modeling an Antibody Figure 9.7. The Antibody Humanization: Residue Mutation panel, Residues tab. 11. Click in the Mutations column for residue L:1 and select None, then press ENTER. The mutations are removed from the row and the text shows None. The mutated structures are refined as part of the mutation job. You can specify the range of the refinement around the mutation and the refinement method. For this exercise, the defaults will be used. 12. Click Run. The job starts, and progress is reported at the bottom of the panel. The job takes about an hour. If you have a multiprocessor host available, you can divide the job into subjobs and distribute them across processors. To do this, click the Settings button and choose the host, the number of processors, and the number of subjobs. The number of subjobs must not be greater than the number of mutations, and must not be less than the number of processors. BioLuminate 1.9 Quick Start Guide 57

64 Chapter 9: Modeling an Antibody When the job finishes, the Humanize Antibody Viewer opens to display the results, and the mutated structures are added to the project. The results viewer lists the mutations in a table, with changes in various properties due to the mutation. It also displays a plot of one of these properties as a function of the table row number. Figure 9.8. The Humanize Antibody Viewer panel. 13. Choose Δ Stability from the Graph property option menu. A plot of the change in stability with respect to the parent structure is displayed. One of the mutations significantly increases the stability, one significantly decreases the stability, and the rest have small or moderate effects. 14. Close the Residue Scanning Viewer panel. 15. Choose File Close Project, and click Discard in the Save Scratch Project dialog box. The project is closed and discarded. 58 Schrödinger Software Release

65 BioLuminate Quick Start Guide Chapter 10 Chapter 10: Protein-Protein Docking This chapter provides exercises that demonstrate how to run protein-protein docking calculations. One protein is treated as the receptor and the other as the ligand. In the general case, it does not matter which protein is treated as the receptor and which protein is treated as the ligand. For antibody-antigen docking, the receptor is the antibody and the ligand is the antigen. The docking is performed as a rigid-body optimization: there is no subsequent minimization of the interfacial region. Two examples are given here: a general docking example, and an antibody-antigen docking example. Structures are taken from the PDB. Protein-protein docking calculations take several hours to run. If the Workspace is not empty, in the Toggle Table panel click the A button in the All row and choose Remove Everything from Workspace General Protein-Protein Docking The receptor used in this example is 1qqu, which is a single chain. The ligand is chain B of 1ba7. The reference structure is 1avx, whose chains differ a little from those of the receptor and the ligand, but not in the binding region. 1qqu differs from chain A of 1avx by two point mutations on the opposite side of the protein from the binding region. Chain B of 1ba7 differs from chain B of 1avx by two small insertions, one in each structure, but not in the binding region. The conformations of chains A and B of 1avx differ a bit from the free proteins due to the formation of the complex. The exercises will compare docking the native ligand, chain B of 1avx, to docking the 1ba7 ligand. As the jobs take 4 hours to run, you should first create a project for the jobs, so that you can close the BioLuminate interface and return to the project later to view and analyze the results, if you want. 1. Choose File Save Project As. 2. Navigate to a location, and enter a name in the File name text box, such as 1avx_docking_exercise. BioLuminate 1.9 Quick Start Guide 59

66 Chapter 10: Protein-Protein Docking Docking the Ligand Protein As Is In this part of the exercise, you will dock the ligand protein to the receptor protein without any modification other than what is done by the Protein-Protein Docking panel. 1. Choose Tasks Protein-Protein Docking. The Protein-Protein Docking panel opens. 2. In the Mode section, check that Standard is selected. 3. In the Protein structures section, click Receptor, then click From PDB ID. 4. Enter 1qqu in the Enter PDB ID dialog box, and click OK. A question box opens, prompting you to add hydrogens to the structure. 5. Click Add Hydrogens. The protein is displayed in the Workspace and the box next to the receptor is filled in with the identity of the receptor. Chain A is selected, as this is the only chain available. The View button next to this box is enabled: this button allows you to view the protein and zoom to it in the Workspace. Figure The Protein-Protein Docking panel with receptor and ligand defined. 6. In the Protein structures section, click Ligand, then click From PDB ID. 7. Enter 1ba7 in the Enter PDB ID dialog box, and click OK. A question box opens, prompting you to add hydrogens to the structure. 60 Schrödinger Software Release

67 Chapter 10: Protein-Protein Docking 8. Click Add Hydrogens. A dialog box opens, prompting you to select the chains to dock. In general, you can choose as many chains as you like: there is no restriction on the number of chains. 9. Select chain B, and click OK. The entire protein is displayed in the Workspace and the box next to the ligand is filled in with the identity of the ligand. The View button next to this box is enabled. The Constraints section is also enabled. In this exercise, no constraints are applied. 10. Click the Settings button. A Job Settings dialog box opens, in which you can choose a host and name the job. 11. Enter 1qqu-1ba7-raw in the Name text box. 12. Ensure that Append new entries is chosen from the menu in the Output section. 13. (Optional) Choose a host. The entire protein-protein docking exercise involves three docking runs, which can be run concurrently. If you have enough licenses, processors, and memory, you can run the job locally. If you have access to a remote host (such as a cluster) you can select that host. The choice you make for the host is the default for the next job. The job takes about 4 hours on a single processor. To reduce the time, you can distribute the job over multiple processors, by selecting a multiprocessor host and entering the number of processors in the Total processors text box. For example, using 4 processors will reduce the turnaround time to about 1 hour. 14. Click Run. The job starts. If you have the resources to run more than one job at a time, you can proceed immediately to the next part of the exercise, where the missing side chains will be added to 1ba7 chain B and the resulting structure will be docked to 1qqu. Otherwise you can proceed when the job finishes Docking the Prepared Ligand Protein In this part of the exercise, you will prepare the ligand protein, chain B of 1ba7, by adding the missing side chains. The protein also has two short loops that could be built, but this exercise does not include building them. 1. In the 1QQU row of the Toggle Table panel, choose A Remove from Workspace. 1qqu is removed from the Workspace, leaving only 1ba7. The side chains will now be added to this structure. BioLuminate 1.9 Quick Start Guide 61

68 Chapter 10: Protein-Protein Docking 2. Choose Tools Protein Preparation. The Protein Preparation Wizard panel opens. 3. Select Remove original hydrogens. 4. Select Fill in missing side chains using Prime. 1. Select this option. 2. Select this option. 3. Click this button. Figure Adding side chains. 5. Click Preprocess. A dialog box opens, asking you to confirm the addition of missing side chains. 6. Click Continue. The structure is processed, removing the hydrogens that were added on import and readding them later, and the side chains are added. The addition of the side chains takes a few minutes. When it is complete, a new entry is added and shown in the Workspace, and you can now use this entry for docking. 7. Close the Protein Preparation Wizard panel. 62 Schrödinger Software Release

69 Chapter 10: Protein-Protein Docking 8. In the Protein-Protein Docking panel, click Ligand, then click From the Workspace. A dialog box opens, prompting you to select the chains to dock. In general, you can choose as many chains as you like: there is no restriction on the number of chains. 9. Select chain B, and click OK. The ligand has been replaced with the new structure that has side chains. 10. Enter 1qqu-1ba7-prep in the Job name text box. 11. Do one of the following: Click Run to start the job on the same host as the previous job. Click the Settings button to choose a different host (see Step 13 in the previous section), and click Run in the Job Settings dialog box to start the job Docking the Native Ligand to the Native Receptor To find out what sort of accuracy can be expected for protein-protein docking, it is a good idea to run a self-docking experiment, if possible. The reference protein 1avx is the complex of two chains that are very close in sequence to the receptor and ligand that we have been using, 1qqu and 1ba7 chain B. The chains of 1avx adopt the optimal conformation in the complex, so if we use chain B of 1avx as the ligand and chain A of 1avx as the receptor, we should get the best possible docking result. This result can be used as a standard for comparison of the results of the two docking runs performed so far. To compare the native ligand (1avx chain B) to the docked poses, the receptor in 1avx (chain A) needs to be aligned to 1qqu, so that they are in the same frame of reference. This is not necessary for the docking run, but only for comparison of the results. The alignment also tells us how closely 1avx chain A matches the receptor protein 1qqu. 1. In the All row of the Toggle Table panel, click the A button in and choose Remove Everything from Workspace. 2. Click Include in Workspace. The Include in Workspace panel opens, listing the titles and IDs of the project entries that you can include. 3. Choose the 1QQU entry and click OK. The receptor structure is now in the Workspace. 4. Choose File Get PDB. The Get PDB FIle panel opens. BioLuminate 1.9 Quick Start Guide 63

70 Chapter 10: Protein-Protein Docking 5. Enter 1avx in the PDB ID text box, and click Download. The reference structure is added to the Workspace, with the receptor in the background. 6. On the main menu bar, choose Tools Protein Structure Alignment. The Protein Structure Alignment panel opens over the Toggle Table. The atoms in the Workspace are marked in yellow. Figure The Protein Structure Alignment panel. 7. Ensure that Use proteins from is set to Workspace (included entries). 8. Click the Clear (X) button in the Reference residues section. The yellow markers are removed. 9. From the Pick menu, choose Chains. 10. Pick an atom in the receptor structure 1qqu (not the reference structure). Yellow markers are added to the receptor structure and chain A of the reference structure. The text chain.name A is added to the ASL text box. 11. Ensure that Use same ASL as reference residues is selected under Residues to align. 64 Schrödinger Software Release

71 Chapter 10: Protein-Protein Docking 12. Click Align. After a short while, the proteins are aligned. The alignment score and the RMSD are reported in the Protein Structure Alignment Results panel. The score should be and the RMSD should be (you might get slightly different results on different systems). 13. Close the Protein Structure Alignment panel and its results panel. The yellow markers are removed. 14. Click the Reset button in the Toggle Table to center the proteins. 15. In the 1QQU row of the Toggle Table panel, choose A Remove from Workspace. 1qqu is removed from the Workspace, leaving only 1avx, which will now be used to set up the self-docking run. 16. In the Protein-Protein Docking panel, click Receptor, then click From the Workspace. A question box opens, prompting you to add hydrogens to the structure. 17. Click Add Hydrogens. A dialog box opens, prompting you to select the chains to dock. 18. Select chain A, and click OK. 19. In the Protein-Protein Docking panel, click Ligand, then click From the Workspace. Chain B is automatically selected because it is the only available chain. 20. Enter 1avx-self-dock in the Job name text box. 21. Click Run. If you want to choose a host, see Step 13 in Section The job starts. If you have worked through these exercises (and are not running other jobs), you should see 3/3 on the Jobs button at the bottom of the Workspace, indicating that three jobs are running out of a total of three. The jobs take about 4 hours each. However, if your resources are limited (either by licenses or by processors), you may see 1/3 on the Jobs button, as the jobs are being run sequentially. Then the total run time will be 12 hours Comparing the Top Poses When the jobs finish, you should restart BioLuminate if you closed the interface, and reopen the project if you closed it (File Open Project or Open Recent Project). When the project opens, you may be prompted to incorporate the results of the jobs, which you should do. In the first part of the analysis, the top poses will be compared to the reference pose. BioLuminate 1.9 Quick Start Guide 65

72 Chapter 10: Protein-Protein Docking 1. In the All row of the Toggle Table panel, choose A Remove Everything from Workspace. This ensures that you are starting with nothing displayed. 2. Click Include in Workspace. 3. Select 1AVX and the three Ligand_ lines, and click OK. The ligand lines should be the first line under each receptor. Make sure that you select them in order raw, then prepared, then self-dock because they all have the same title, and you will not otherwise be able to tell which is which. The reference structure and the best ligand pose for each docking run is included in the Workspace. The Workspace looks a bit cluttered, but we will look at each pose in turn. 4. In the All row of the Toggle Table panel, choose S As Cartoon. The structures are shown in cartoon representation. This makes visual comparison of the poses easier: showing the atoms gives too much detail. 5. In the 1AVX row of the Toggle Table panel, choose C Color by Chain by Chain. The reference structure chains are colored, which distinguishes them from the ligands. 6. Click the rows in the Toggle Table panel for the first and second ligands. These ligands are hidden, and you can now see the reference ligand and the pose from the self-docking run. 7. Rotate the structure to examine the match between the reference and the docked pose. The docked pose is displaced and rotated somewhat from the reference. This is due to the docking algorithm, because the pose should be exactly over the reference ligand if the docking were perfect. This shows the level of accuracy you can expect from docking. Figure Docked poses (gray) with the 1avx protein (receptor green, ligand blue). From the left: raw, prepared, self-docked. 66 Schrödinger Software Release

73 Chapter 10: Protein-Protein Docking 8. Click the rows in the Toggle Table panel for the second and third ligands. The second ligand is shown and the third is hidden, and you can now see the reference ligand and the pose from the docking run with the prepared ligand. 9. Rotate the structure to examine the match between the reference and the docked pose. The agreement is similar to that for the self-docked ligand. 10. Click the rows in the Toggle Table panel for the first and second ligands. The first ligand is shown and the second ligand is hidden, and you can now see the reference ligand and the pose from the docking run with the raw ligand. 11. Rotate the structure to examine the match between the reference and the docked pose. The agreement is not as good as that for the prepared ligand. This is to be expected, because there are missing side chains in the binding region Calculating the RMSD Between the Top Poses To quantify the difference, the RMSD between the docked ligands and the reference ligand can be calculated. To avoid problems with missing side chains in the raw pose and sequence identity, the RMSD will be calculated between the alpha carbons of the first 124 residues. 1. Choose Tools Superposition. The Superposition panel opens below the Toggle Table. 2. Ensure that Included entries is selected under Entries to superimpose. 3. Select Calculate 'in place' (no transformation). This choice just calculates the RMSD without superimposing the structures. 4. Select Create RMSD property. 5. In the ASL tab, click Select. The Atom Selection dialog box opens. 6. In the Chain tab, choose B from the Chain name list, and click Add. 7. In the Atom tab, choose PDB type from the list on the left. The list of PDB atom types is displayed in the center, with the heading PDB type. 8. Choose CA from the PDB type list, and click Intersect. BioLuminate 1.9 Quick Start Guide 67

74 Chapter 10: Protein-Protein Docking Figure The Atom Selection dialog box for superposition of poses. 9. In the Residue tab, choose Residue number from the list on the left. 10. In the Residue number text box, enter the text 1-124, , and click Intersect. The reason for the two residue ranges is that the reference residue chain B is numbered starting at 501 rather than at Click OK. The calculation of the RMSD is performed, and the results appear in the RMSD text box. They are also added to the project as a property. The RMSD values are about 4.7 for the self-docked pose, 5.4 for the prepared ligand pose, and 8.8 for the raw ligand pose. The difference between the self-docked pose and the prepared ligand pose is relatively small, and could possibly be explained mainly by the fact that the ligand from 1ba7 does not superimpose exactly on the reference ligand, even with the restriction to the common part of the chain. The RMSD for doing this superposition is about 0.5. (You can verify this by including only the reference ligand and the prepared ligand, and deselecting Calculate 'in place' in the Superposition panel, so that the ligands are actually moved.) The difference between the raw ligand and the prepared ligand poses, on the other hand, is significant, and demonstrates the importance of preparing the proteins. Many proteins have 68 Schrödinger Software Release

75 Chapter 10: Protein-Protein Docking missing side chains or loops on their surface, because these regions are more mobile and are more likely to be poorly or incompletely modeled in the PDB structure. Using the Protein Preparation Wizard and adding both side chains and filling short loops with Prime is recommended. If the X-ray data is available, you can consider using PrimeX to obtain a better structure, guided by the density. Long loops can also be modeled by Prime, but in the absence of structural information it might be better to disregard poses that include long missing loops in your docking runs. 12. Close the Superposition panel Examining the Poses and the Binding Sites The final part of this exercise is to examine the poses that are generated, to see where on the protein the ligand is likely to dock. For this purpose, it is better to use the Project Table, which enables you to easily step through the display of a set of entries in the Workspace. 1. Choose File Project Table, or press CTRL+T ( T). The Project Table panel opens. 2. Click the row that contains 1qqu-1ba7-raw in the title. This row should have a number in square brackets in the Title column. It is the row for an entry group, which is a collection of project entries. This group contains the docking results for the raw run. 3. Choose Entry Include Only. After a short while, all the entries in the group are included in the Workspace. 4. In the All row of the Toggle Table, choose S As Cartoon. The entries are all changed to cartoon representation. 5. In the Project Table panel, choose Entry Exclude. The entries are removed from the Workspace again, but they are now set up in cartoon representation so that they can be compared. 6. Click the + sign in the In column for the group row. The group is expanded so you can see all the entries. All of the rows are highlighted in yellow to show that they are selected. 7. Select the receptor row. The receptor row is now the only row highlighted. BioLuminate 1.9 Quick Start Guide 69

76 Chapter 10: Protein-Protein Docking 8. Choose Entry Fix. The receptor entry is included in the Workspace, as indicated by the box in the In column. There is also a padlock icon, to indicate that this entry will remain in the Workspace until it is explicitly removed. 9. Zoom out in the Workspace so that you can see the receptor and enough space around it to fit a ligand of the same size. Click the group row again, to select all its entries. This is the row that contains 1qqu-1ba7-raw in the title. 10. With the pointer in the Workspace, press the RIGHT ARROW key. The first ligand is displayed in the Workspace. 11. Press the RIGHT ARROW key repeatedly, to display the ligands in turn. To go back, you can press the LEFT ARROW key. The majority of the ligands bind in the native binding site, but a few bind in other sites. It is clear that there are a few sites that favor binding, and other regions that do not favor binding. This concludes the exercise, and you can now clean up. 12. In the All row of the Toggle Table panel, choose A Remove Everything from Workspace. 13. Close the Protein-Protein Docking panel, if it is still open. 14. Choose File Close Project Antibody-Antigen Docking This exercise uses the antibody-antigen docking mode, in which the non-cdr regions of the antibody are automatically detected and masked in the docking. The reference for this exercise is 1fsk: the bound antigen is chain A and the antibody consists of chains B (the light chain) and C (the heavy chain). This exercise uses chains B and C for the receptor. The free antigen is 1bv1, which is used for the ligand, and has the same sequence as chain A of 1fsk. The RMSD for superposition of the free antigen on the bound antigen is 0.65 angstroms. First, save the project so that you can come back to it later. The docking run takes over 5 hours on a single processor. 1. Choose File Save Project As. 2. Navigate to a location, and enter a name in the File name text box, such as antibody_docking_exercise. 70 Schrödinger Software Release

77 Chapter 10: Protein-Protein Docking Preparing the Antibody The 1fsk structure is actually a tetramer, with 12 chains, so a single antibody-antigen unit will be extracted for use in this exercise. A convenient way of doing this is to use the Protein Preparation Wizard panel, even though the antibody does not need processing for this exercise. 1. Choose Tools Protein Preparation. 2. Enter 1fsk in the PDB text box, and click Import. The 1fsk structure is imported into the project and displayed in the Workspace. 3. In the Review and Modify tab, click Analyze Workspace. The chains are listed in the table. 4. Select chains A, B, and C. 5. Click Invert Selection, then click Delete. Chains D and higher are deleted, leaving chains A, B, and C. This is the reference structure that will be used for comparing the results, and also for the antibody receptor. 1. Click this button. 4. Click this button. 2. Select chains. 3. Click this button. Figure Deleting chains. BioLuminate 1.9 Quick Start Guide 71

78 Chapter 10: Protein-Protein Docking 6. Close the Protein Preparation Wizard panel Setting Up and Running the Job With the reference structure imported, you can now set up the docking run. 1. Choose Tasks Protein-Protein Docking. The Protein-Protein Docking panel opens. 2. In the Mode section, select Antibody. The Mask non-cdr region option is now available, and is selected by default. This option restricts the docking to the CDR region. For this exercise, the option should remain selected. 3. In the Antibody structures section, click Receptor, then click From the Workspace. A question box opens, prompting you to add hydrogens to the structure. 4. Click Add Hydrogens. The antibody is imported and analyzed to determine which are the light and heavy chains, and to locate the CDR regions. This operation takes a minute, and a progress bar is displayed at the bottom of the panel. If you had imported 1fsk directly, the analysis would have taken four times as long, so it is worth trimming down the structure beforehand. When the analysis finishes, a dialog box opens, prompting you to select the chains to use for the antibody receptor. You must select two chains: a light chain and a heavy chain. 5. Select chains B and C, and click OK. The text box for the antibody is filled in with the information on the structure used. 6. Click Antigen, then click From PDB ID. 7. Enter 1bv1 in the Enter PDB ID dialog box, and click OK. An alert box opens, warning you about the chains. You can dismiss this box. 8. Click the Settings button. The Job Settings dialog box opens, in which you can choose a host and name the job. 9. Ensure that Append new entries is chosen from the menu in the Output section. 10. Enter 1fsk-1bv1 in the Name text box. 11. (Optional) Choose a host and set the number of processors. The job takes about 5 hours on a single processor, so distributing it is recommended. 72 Schrödinger Software Release

79 Chapter 10: Protein-Protein Docking 12. Click Run Comparing the Best Pose with the Reference After the results are imported, you can compare the best pose with the reference structure. Both a visual and a quantitative comparison are done in this section. 1. Remove everything but 1fsk from the Workspace. You can do this with A Remove from Workspace for each row. 2. In the 1FSK row, choose C Color by Chain by Chain. The three chains are colored with different colors. 3. Click Include in Workspace. 4. Select Antigen_ and click OK. The first (best ranked) antigen pose is included in the Workspace. 5. In the All row of the Toggle Table, choose S As Cartoon. The proteins are shown in cartoon representation. You can see that the docked pose matches the native pose very well. To quantify the agreement, the RMSD will be calculated between the alpha carbons. Figure The 1fsk reference structure with the docked antigen pose (in gray). BioLuminate 1.9 Quick Start Guide 73

80 Chapter 10: Protein-Protein Docking 6. Choose Tools Superposition. The Superposition panel opens below the Toggle Table. 7. Ensure that Included entries is selected under Entries to superimpose. 8. Select Calculate 'in place' (no transformation). This choice just calculates the RMSD without superimposing the structures. 9. In the ASL tab, click Select. The Atom Selection dialog box opens. Figure The Atom Selection dialog box for antigen superposition 10. In the Chain tab, choose A from the Chain name list, and click Add. 11. In the Atom tab, choose PDB type from the list on the left. The list of PDB atom types is displayed in the center, with the heading PDB type. 12. Choose CA from the PDB type list, and click Intersect. 13. Click OK. 74 Schrödinger Software Release