1. Double-click the BioLuminate icon.
   • (No icon? See Starting Maestro)

 

Figure 1-1. Change Working Directory option.

2. Go to File > Change Working Directory.
3. Find your directory, and click Choose.
4. Pre-generated input and results files are included for running jobs or examining output. Download the zip file here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/peptidemodeling_bl.zip
5. 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 1-2. Opening the project.

6. Go to File > Open Project and choose peptidemodelingtutorial.prjzip.
7. Click Open.
   • Structures are shown in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

 

Note: Please see the Glossary of Terms for the distinction 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.

 

8. In the Save scratch project warning box, click OK.

Figure 1-3. Saving the project via the Save Project panel.

9. Go to File > Save Project As.
10. Change the File name to peptidemodeling_new.
11. Click Save.
    • The project is now named peptidemodeling_new.prj.    

Figure 2-1. Including 1AWR_complex_prep entry and selecting 1AWR-random entry.

For this retrospective validation, we would want to start with a randomized input conformation of the peptide such that we can show that the docking algorithm is able to generate the crystallographic pose.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue 1AWR_complex_prep in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
   • A receptor structure is loaded into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
2. 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 1AWR-random in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
3. Double-click Presets.
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is rerendered in the default Custom Preset.

Figure 2-2. Peptide Docking in Biologics Tasks.

 

4. Go to Tasks > Biologics > Peptide Docking.

Figure 2-3. The Peptide Docking Panel.

5. In the Binding Site section, check the Pick box for Centroid option.
   • A banner appears prompting you to pick a ligand to define the region.

Figure 2-4. Receptor bounding boxes.

6. Select any atom of the ligand in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
   • The ligand is now highlighted with a purple box and green box around it.
   • The ligand will be excluded from the grid generation.

 

Note: The purple bounding box defines the region that the docked molecule(s) can occupy to satisfy the initial stages of docking. The green box defines where the midpoint of any docked molecule must reside to satisfy the initial stages of docking.

Figure 2-5. Adding selected sequences from the Project Table.

7. For Add Sequences from, choose Project Table.
   • The 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 1AWR-random is displayed in the Peptide table.
8. Change the Job name to 1AWR_pep_MMGBSA.
9. Click Run.
   • This job will take ~1 hour to run on a single CPU.
   • To save time, you will look at the pregenerated results.

Figure 2-6. Selecting the 1AWR_pep_MMGBSA group for viewing poses.

 

 

1. 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 the group 1AWR_pep_MMGBSA in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
2. Right-click on the group header and select View Poses.
   • The Pose Viewer panel opens.
   • The first structure in the group is fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, the second is includedthe entry is represented in the Workspace, the circle in the In column is blue.

 

Figure 2-7. Pose Viewer Set Up.

3. Ensure Display per-residue interactions is disabled.
4. Click Set Up Poses.
5. Step through the results using the right and left arrow keys.

 

Note: Turn off AUTO-fit on the main toolbar to prevent the Workspace from zooming to fit the selection when viewing ligands.

 

Figure 2-8. Overlay of crystallographic ligand (green) with the 18th docked pose (cyan).

5. Double-click the In circle to fix 1AWR_chainG in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
6. Compare poses to the cognate liganda ligand that is bound to its protein target using the right and left arrow keys.
7. Close the Pose Viewer panel.

Figure 3-1. The MM-GBSA Residue Scanning Calculations option in Biologics Tasks.

1. Go to Workspace > Clear Workspace.
2. Includethe entry is represented in the Workspace, the circle in the In column is blue 2NL9_prepped in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: Type Z to fit the structure to the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, if needed.

 

3. Go to Tasks > Biologics > MM-GBSA Residue Scanning Calculations.

Figure 3-2. Calculating for Stability and Affinity of Chain A binding to Chain B.

4. For Import structure from, choose Workspace.
5. Click Import.
   • The Residues table gets populated.
6. For Calculation type, choose Stability and Affinity.
7. For Binding partners, uncheck the B checkbox in the Chains menu.
   • Chain A is binding to Chain B.

Figure 3-3. Mutating selected residues to ALA.

8. For Set mutations for, choose Selected residues.

 

To evaluate the residue at the peptide-protein interface, you will use alanine scanning to predict the impact on both affinity and stability that would arise when an amino acid on the interface has its side chain mutated to an alanine side chain.

 

9. In the table, use Ctrl+Click (Cmd+Click) to 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 the following residues of chain B: 54-58, 60, 62-70, 72-73.
10. Next to ALA, click Apply.
    • 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 mutations are set to ALA.

 

Note: It has been documented that there is a bias toward arginine and methionine in residue scanning outputs. It is recommended to keep this in mind when designing your residue scanning experiments, and to look critically at results that suggest beneficial methionine or arginine mutations.

Figure 3-4. Opening the Nonstandard Residues panel.

11. Go to the Options tab.
12. Click Define Nonstandard Residues.
    • The Nonstandard Residues panel opens.

 

Figure 3-5. Managing the Nonstandard Residues.

Note: The Nonstandard Residue panel is loaded with all standard amino acids and a selection of 48 nonstandard amino acids.

 

13. Find DGL in Row 35 and check Mutate to checkbox.
    • Mutating to DGL is now an option in the MM-GBSA Residue Scanning panel.

Figure 3-6. Selecting residues to mutate to GLU and DGL.

17. Go to the Residues tab.

 

To explore the alanines to the peptide-protein interface, you are going to mutate them to the much-bulkier GLU and DGL side chains.

 

18. Mutate residues 59, 71, and 74 of chain B to GLU and DGL.
19. Change the Job name to mcl1_bim_alaninescan.
20. Click Run.
    • These calculations take ~30 seconds per mutation.
    • To save time, you will look at the pregenerated results.

Figure 3-7. Loading the MM-GBSA Residue Scanning results.

21. Go to Tasks > Biologics > MM-GBSA Residue Scanning Results.
    • The MM-GBSA Residue Scanning Viewer panel opens.
22. Next to Load results from, choose File, and click Browse.
23. Select mcl1_bim_alaninescan-out.maegz.
24. Click Open.
    • The Mutations table is populated with the predicted changes in affinity and stability.

Figure 3-8. Adjusting the graph results.

25. In the Graph representation, select the area of the graph to cover Δ Affinity of 15 by clicking and dragging in the graph area.
26. Check Sync selection with mutations table.
    • Filtered results are now displayed in bold and selected in the Mutations table.

Figure 3-9. Adjusting the Workspace Display.

27. In Mutations, click Workspace Display and check Original residue in gray.
28. Select Residue B:62.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is zoomed to residue 62.
    • The mutation is green, while the original LEU residue is gray.

Figure 3-10. The LEU62 (gray) mutation to alanine (blue).

Note: In this example, alanine mutations with large, positive values for Δ Affinity are interpreted as being potential binding hot spots.

Figure 4-1. Include 1BXL_minimized in the Workspace.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue 1BXL_minimized in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
2. Go to Tasks > Biologics > MM-GBSA Residue Scanning Calculations.

Figure 4-2. Calculating for Stability and Affinity of Chain A binding to Chain B.

3. For Import structure from, choose Workspace.
4. Click Import.
   • The table is populated.
5. For Calculation type, choose Stability and Affinity.
6. For Binding partners, uncheck the B checkbox in the Chains menu.
   • Chain A is binding to Chain B.

Figure 4-3. Selecting residues to mutate to NonPolar (Hydrophobic) residues.

7. For Set mutations for, choose Selected residues.
8. In the table, use Ctrl+Click (Cmd+Click) to select the following residues of chain B: 574, 578, 581, and 585.
9. Click the second menu for Set mutations for (following “to”), and check NonPolar (Hydrophobic).
10. Click Apply.
    • Selected mutations are set to nonpolar residues.

Figure 4-4. Options tab of the MM-GBSA Residue Scanning panel.

11. Click the Options tab.
12. Check Perform affinity maturation (protein design).
    • The Combinations section appears.
13. For Property to optimize, choose Affinity.
14. Change Job name to bclxl_bak_affinitymaturation.
15. Click Run.
    • This job will take ~4 hours to run on a single CPU.
    • To save time, you can use the pregenerated results.

Figure 4-5. Viewing the affinity maturation results.

1. Go to Tasks > Biologics > MM-GBSA Residue Scanning Results.
   • The MM-GBSA Residue Scanning Viewer panel opens.
2. Next to Load results from, choose File.
3. Click Browse and locate the bclxl_bak_affinitymaturation-out.maegz file.
4. Click Open.
   • Results are displayed in the table and graph.
   • Each row of data corresponds to a combination of mutations.

Figure 4-6. The resulting LOGO plot.

5. Click Create LOGO plot at the bottom of the panel.
   • A sequence LOGO plot opens.
   • A .png image of the LOGO plot is saved to your Working Directorythe location where files are saved.
6. Click OK.

 

Note: Given the known bias toward methionine, these affinity maturation results should be examined more carefully.

Figure 5-1. Peptide QSAR in Biologics Tasks.

1. Go to Tasks > Biologics >  Peptide QSAR.
   • The Peptide QSAR panel opens in the Setup tab.

Figure 5-2. Loading peptide sequences.

2. Click Load Sequences and Observables.
   • The Peptide QSAR - Load Sequences and Observables panel opens.
3. For Load sequences from, choose CSV file.
4. Click Browse.
5. Choose TIAN.csv and click Open.
6. Click OK.
   • A dataset of 153 peptide sequences is loaded into the Peptide QSAR table.

Figure 5-3. Setting Peptide descriptor type and Maximum number of PLS factors.

7. For Peptide descriptor type, choose dpps.
8. Click Advanced Options for PLS.
9. Set the Maximum number of PLS factors to 10.
10. Click OK and then click Build.
    • When the model is built, the Results tab opens.

 

Note: The more PLS factors used to build the model, the more likely the model is to be over-fitted. As a suggestion, set the maximum number to be no more than the number of sequences divided by ten.

Figure 5-4. Results for the peptide QSAR model.

Note: While the Pearson correlation coefficient (R^2) for the Training set is satisfactory, the corresponding statistic for the Test set (Q^2) is significantly worse.

 

11. Next to PLS factors, click the up and down arrows.
    • The statistics don’t change much with altering the number of PLS factors.
    • This suggests that a smaller number of factors could be used to build a comparably predictive model.
12. Click Plot.
    • Peptide QSAR Scatter Plot panel opens showing Observed vs. Predicted activity values for the test and training sets.

Figure 5-5. Scatter Plot of Observed vs. Predicted activity values for the test and training sets.

Note: While the statistics could be improved, a trend is observable in the data, with only a few large outliers. However, to increase the robustness of the model, more data is necessary. Try decreasing the percentage of the dataset allocated to Test to 25% and you will observe that while R^2 increases significantly, Q^2 suffers, suggesting that the model is overfit.

 

13. Close the Peptide-QSAR Scatter Plot.

Figure 5-6. Changing to 25% of the dataset allocated for the test set.

14. Go to the Setup tab.
15. Next to Use all rows in the table. Randomly select 25% for the test set.
16. Click Build.

Figure 5-7. Results of overfit model.

17. Go to the Results tab.

 

Note: With a 25% test set, while R^2 has increased slightly, Q^2 is much lower. This suggests that the model is overfit.