1. Open BioLuminate and create a new project named antibody-antigen_docking.prj for this tutorial.
   • Don’t know how? See First steps in Maestro.
2. Download the tutorial zip file including input files and reference outputs here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/antibody_antigen_docking.zip
3. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

Figure 2-1. Importing the structure from the PDB.

4. Go to File > Get PDB.
   • The Get PDB File dialog box opens.
5. For PDB IDs, type 1E6J.
6. Click Download.
   • A banner appears and the structure is loaded into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Note: Imported structures in Maestro are includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion by default. Please refer to the Glossary of Terms for the difference between includedthe entry is represented in the Workspace, the circle in the In column is blue and selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

Figure 3-1. Specifying the structure for preparation in the Protein Preparation Workflow panel.

1. Click Prepare in the banner or click Protein Preparation in the Favorites toolbar.
   • The Protein Preparation Workflow panel opens in the Preparation Workflow tab.
2. In the Specify Protein section, for Use structures from, choose Workspace (included entry).

Figure 3-2. Viewing the issues in the Diagnostics tab.

Before preparing the structure, you should review potential issues.

3. Go to the Diagnostics tab.
4. Click Check Workspace Entry.
   • Valence errors are present in the structure.
   • These are caused by crystallographically invisible Hydrogen atoms and will be resolved during protein preparation.

Figure 3-3. Reviewing the structure before preparation.

You can also review the contents of the structure. This is an easy way to remove crystallographic artifacts or some of the chains (and ligands or solvents associated with them) if you need to.

5. Go to the Substructures tab.
6. Click Load Workspace Entry.
   • The Chains table populates.
   • Chain H and L belong to the murine antibody (Heavy and Light chains respectively) and the chain P is the HIV-1 capsid protein p24 (antigen).

Note: There are no crystallographic artifacts and solvent molecules in the 1E6J structure.

7. Click Workflow > at the bottom to return to the Preparation Workflow tab.

Figure 3-4. Modifying Preprocess settings.

8. Under Preprocess, click More Options.
9. Confirm Identify Protein Features is checked and Antibody is selected.
10. For Antibody scheme, choose Chothia.

Figure 3-5. Running the preparation job.

The other default settings work well for the chosen system. For an in-depth understanding of various options in the Protein Preparation Workflow, see the Introduction to Structure Preparation and Visualization tutorial and the panel documentation.

11. Change the Job name to proteinprep_1E6J.
12. Click Run.
    • This job takes ~ 1 minute.
    • Once the job is completed, a banner appears and a new group proteinprep_1E6J-out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
13. Close the Protein Preparation Workflow panel.

Note: If your structure contains crystallographic solvents, we recommend duplicating the prepared structure and deleting all solvents from the copy before proceeding with docking.

Figure 4-1. Applying presets to the prepared structure.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue 1E6J – prepared in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
2. Click Presets.
3. Choose Antibody > Chothia.
   • The complex structure is rendered in cartoon-styled ribbons.
   • The blue and red colored chains are the heavy and light chains of the antibody respectively, while the green colored chain is the antigen.

Note: You can hover over the chains to see their corresponding names in the Status Bar.

Figure 4-2. Copying antigen chain to a new entry.

You will now create separate entries for the antibody and the antigen for performing the docking calculation.

4. In the Hierarchy, expand Protein.
5. Select Chain P.
6. Right-click the selection and choose Copy to New Entry.
   • A new entry is created and added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
   • A banner appears.
7. Rename the newly created entry to 1E6J_antigen.

Figure 4-3. Copying antibody chains to a new entry.

8. Includethe entry is represented in the Workspace, the circle in the In column is blue 1E6J – prepared in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed again.
9. In the Hierarchy, expand Protein and then expand Antibody.
10. Ctrl+Click (Cmd+Click) to select chain L (LC) and Chain H (HC).
11. Right-click the selection and choose Copy to New Entry.
12. Rename the newly created entry to 1E6J_Fab.

Note: You can also double-click any entry title to rename it.

Figure 4-4. Opening the Protein-Protein Docking panel.

13. Includethe entry is represented in the Workspace, the circle in the In column is blue 1E6J_Fab in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries 1E6J_antigen in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
14. Find and open the Protein-Protein Docking panel from Tasks.

Figure 4-5. Specifying the antibody to dock.

You will perform docking in the Antibody mode.

15. For Mode, choose Antibody.
16. Confirm Mask non-CDR region is checked.
    • This ensures that the attractive potential for residues in the non-CDR region is removed. The Chothia definition is used for the CDR region.
17. In the Structures to Dock section, click Antibody option menu and choose From the Workspace.
    • The Multiple Chains Found dialog box appears.
18. Shift-click to select both H (Heavy) and L (Light) chains.
19. Click OK.

Figure 4-6. Specifying the antigen to dock and running the antibody-antigen docking job.

20. Click Antigen option menu and choose From Project Table (selected entry).

Note: The Refine output poses option, which is selected by default, helps reduce clashes between the two proteins.

By default, PIPER retains the top 1000 scoring poses and clusters them based on the RMSD between matching atoms in each pair. Typically, only the largest ~ 30 clusters are returned, as they are most likely to include near-native conformations. From each cluster, a representative pose is selected – the one with the most neighbors in the cluster.

In this tutorial, we will analyze the representative docked poses from each cluster.

21. Uncheck Preserve archive of top 1000 unclustered poses.
22. Change the Job name to 1E6J_docking.
23. Optional: Click Run.
    • We do not recommend running this job as it takes a long time to run.
    • To save time, the pre-generated results are included in the tutorial files.

Figure 5-1. Visualizing the docked poses.

1. Double-click the In-circle of 1E6J – prepared.
   • The entry is pinned which implies it is fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Now, you will import the output file and compare the representative poses with the prepared crystal structure.

2. Go to Files > Import Structures.
3. Navigate to and choose 1E6J_docking-out.maegz from the tutorial files in your Working Directorythe location where files are saved.
   • A new entry 1E6J_docking-out with 30 poses is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
   • The top entry is included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
4. Use the right and left arrow keys to step through the poses and compare them with the crystal structure.
   • Out of all the 30 poses, 1E6J_docking_pose_1 fits best to the crystal structure.

Figure 5-2. Choosing to perform Protein Interaction Analysis.

5. Right-click the In-circle of 1E6J – prepared entry and Exclude it from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
6. Includethe entry is represented in the Workspace, the circle in the In column is blue 1E6J_docking_pose_1 in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
7. Click the workflow icon next to 1E6J_docking-out group and choose Analysis > Protein Interaction Analysis.
   • The Protein Interaction Analysis panel opens.

Figure 5-3. Performing Protein Interaction Analysis to identify key interactions in one of the docked poses.

8. Under Interaction sets section, for Define sets by option, choose Chain.

The interactions are evaluated between two user-specified atom sets representing the antibody and the antigen.

9. For Set 1, click Add and select chains H and L (of antibody).
10. For Set 2, click Add and select chain P (of antigen).

Note: You can customize the distance thresholds for the interactions using the “Advanced Options” depending on the context of analysis.

11. Click Calculate.
    • This calculation takes a few seconds.
    • The Interactions table populates.
12. Click on the Distance heading to sort the table by distance.

Figure 5-4. Visualizing interactions in the Workspace.

13. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, right-click the Interactions Toggle and confirm the Non-covalent bonds are toggled on.
14. Set the interactions to show antibody-antigen interactions.  
15. In the panel, click on a table row to visualize the corresponding interactions in the 3D Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • Take your time to scroll and view the interactions in the Specific Interactions column. Additionally, scroll to the right or resize the window to view other column results.
16. Optional: Click Diagram to see the Protein Interaction Analysis Diagram.
17. Close the Protein Interaction Analysis panel once you finish visualizing the interactions.

Figure 5-5. Choosing to generate Interaction Fingerprints.

18. Confirm the 1E6J_docking-out group is selected in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
19. Click the workflow icon next to it and choose Interaction Fingerprints.
    • The Interaction Fingerprints panel opens in the Fingerprint Generation tab.

Figure 5-6. The Interaction Fingerprints panel.

You first need to define the interactions you are going to examine.

20. For Generate fingerprints from Project Table (30 selected entries), choose Protein-protein complexes.
21. For Analyze interactions of receptor chains, uncheck P.
    • Chains H and L of the antibody are treated as the receptor and chain P is treated as the ligand.
22. For Interactions to include, select Any Contact.
23. Click Generate Fingerprints button.
    • It takes a few seconds to generate the interaction fingerprints.

Figure 5-7. The Interaction Matrix.

The interaction matrix allows you to better visualize the antibody-antigen interactions found in the docked poses.

24. Click Display Interaction Matrix button.
    • The Interaction Matrix dialog box opens.

You can simplify the matrix by only showing the residues that form at least one interaction.

25. Check Interacting residues only.
    • The plots update.

Note: You can hover over the plots to see the related information.