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/intro_bioluminate.zip
5. After downloading the zip file, unzip the contents in your Working Directory for ease of access throughout the tutorial

 

Figure 1-2. Open Project.

6. Go to File > Open Project
7. Choose BioLuminate_basics.prjzip
8. Click Open
   • Structures are now shown in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Figure 2-1. The Protein Preparation Workflow in the Favorites Toolbar and 3B7E_chainA included.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue entry 3B7E_chainA
2. Click Protein Preparation in the Favorites Toolbar
   • The Protein Preparation Workflow opens

 

Note: The Protein Preparation Wizard can also be accessed by going to Tasks > Browse All > Protein Preparation and Refinement > Protein Preparation Workflow

 

Note: Click on the star next to items in Tasks to add it to the Favorites Toolbar

Figure 2-2. Review Structure.

3. Under Specify Protein, click Review Structure
   • The Substructures tab opens

Figure 2-3. Substructures tab in the Protein Preparation Workflow.

Use the Substructures tab to remove unwanted items from the structure, such as chains, waters, or crystallization agents (e.g. GOL)

 

4. Shift-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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the GOL (Glycerol) rows in the Ligands, Metals, Other table
5. Click Delete from Entry

Figure 2-4. Diagnostic tab in the Protein Preparation Workflow.

6. Click Diagnostics
   • The Diagnostics tab opens

 

Note: The only issues found for the structure is that some of the residues and waters have alternate positions

 

7. Select the 2 residues and 4 waters and click Commit

Figure 2-5. Preparation Workflow.

 

8. Click Preparation Workflow
   • The Preparation Workflow Tab opens
9. For Job name type proteinprep_3B7E_A
10. Click Run
    • This step takes ~5 minutes
    • A new entry is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

 

Figure 2-6. The Presets dropdown.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue entry 3B7E_chainA_prepared
2. Click Presets
3. Choose BioLuminate Default
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is rendered to show the protein as a Green ribbon with the small molecule in grey

 

Note: The Toggle Table on the right of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed can also be used to change rendering. The Toggle Table mimics PyMOL functionality.

 

Note: More information on Workspacethe 3D display area in the center of the main window, where molecular structures are displayed visualization can be found in the Introduction to Structure Preparation and Visualization

Figure 2-7. Surface Binding Site option.

1. Go to Workspace > Surface > Binding Site
   • The Create Binding Site Surfaces panel opens

Figure 2-8. Create Binding Site Surfaces panel.

 

2. Unselect Ligand
3. Click OK
   • A surface is shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
   • Negative regions are red and positive regions are blue
   • An S icon is next to the entry in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Figure 2-9. Electrostatic potential surface.

4. Type L
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to the ligand

 

Figure 2-10. Surfaces toggle in the Workspace Configuration Toolbar.

1. Type M
   • Selection mode is changed to Molecule
   • The cursor has an M icon
2. In the Workspace Configuration Toolbar, turn off Surfaces

Figure 2-11. Expand selection options.

3. Click on the ligand in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
   • The ligand is highlighted
   • The (Selection) entry appears in the Toggle Table
4. Click Expand and choose 5Å
   • Residues within 5 Å of the ligand are highlighted

 

Note: This can also be done using the Toggle Table (Selection) entry A > Modify > Expand > Residues within 5A

Figure 2-12. Show as Sticks in the Toggle Table.

5. In the Toggle Table, for (Selection), choose S > Sticks
   • The selection is rendered in sticks
6. In the Toggle Table, for (Selection), choose   H > Surfaces and H > Waters
   • The surface and waters are hidden
7. In the Toggle Table, for (Selection), choose L > Residues
8. Residue information is labeled in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 2-13. Clear selection in the Quick Select toolbar.

9. In the Quick Select toolbar, click Clear Selection
10. Click on the ligand

Figure 2-14. The Workspace after Toggle Table rendering.

11. In the Toggle Table, for (Selection), navigate to A > Polar Contacts > Find > To Any Atoms Excluding Solvent
    • Hydrogen bonds are shown in yellow, salt bridges in pink

Figure 2-15. Include 3B7E_chainA_prepared and 3BEQ_chainA.

1. Shift-click to includethe entry is represented in the Workspace, the circle in the In column is blue the entry 3BEQ_chainA - prepared (3B7E_chainA - prepared should already be included)
   • Both the drug-bound (green) and apo structures (blue) are visualized in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 2-16. Protein Structure Alignment in the Protein Preparation and Refinement Task panel.

2. Go to Tasks > Browse All > Protein Preparation and Refinement > Protein Structure Alignment
   • The Protein Structure Alignment panel opens on the right side of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, along with a banner
   • By default, all residues are chosen for alignment

Figure 2-17. Protein Structure Alignment panel.

3. Click Align
   • The structures are aligned
   • The Protein Structure Alignment Results panel opens, showing an Alignment Score and RMSD

 

To more easily compare the two structures, we will want to render them identically (though the very low RMSD should already indicate that they will be exceedingly similar).

 

4. Click Presets
5. Choose Binding Site Comparison
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is rendered in a way that you can easily compare the binding site across the two structures

 

Note: This will undo some of the previous changes we made to the visualization of the the 3B7E_chainA - prepared structure

Figure 2-18. Aligned protein structures with 150 loop region highlighted.

6. Hover over the structures to identify regions of interest
   • The 150 loop region has a different conformation between the drug-bound and apo structures
7. Go to Workspace > Save Image As
   • The Save Image panel opens
8. Change File name to aligned_structures
9. Click Save
   • A .png image is saved to your Working Directorythe location that files are saved

 

Note: Click Options >> in the Save Image panel to adjust image properties

Figure 3-1. Multiple Sequence Viewer/Editor in Biologics.

1. Go to Workspace > Clear Workspace
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and Toggle Table are empty
2. Go to Tasks > Biologics > Multiple Sequence Viewer/Editor
   • The Multiple Sequence Viewer/Editor panel opens

Figure 3-2. Import Sequences in the Multiple Sequence Viewer/Editor.

3. Go to File > Import Sequences from File
4. Choose neuraminidase_InfluenzaA_ PDBsequences.fasta
5. Click Open
   • 54 sequences are now in the MSV

 

Figure 3-3. Edit coloring scheme.

To better understand the conservation of residues compared to the reference (which by default is the top sequence) we are going to try some styling that will point out areas where there is variation and areas that are more conserved. This will be more useful after we perform the multiple sequence alignment

 

6. Hover over the brush icon and click the …
7. For Apply to, choose Residues matching reference

Note: By default, the top sequence will be considered as the reference

Figure 3-4. Perform multiple sequence alignment.

8. Click Align

Note: Make sure Selected only is unchecked

9. Click Align
   • Sequences are aligned  

Figure 3-5. Unwrap sequences.

10. Click the plus icon
11. Deactivate Wrap sequences
    • All sequences can now be found on a single row
12. Scroll to the right of the sequence viewer to look at the sequences starting with residue 84

Figure 3-6. Add Logo Plot.

In addition to the styling, we would want to use a Sequence Logo chart to better visualize and (when hovering over) quantify the variation at different positions.

 

13. Hover over the graph icon and click the …
14. Select Sequence Logo
    • A logo plot is added above the reference sequence
15. Click Sequence Logo again to toggle it off

Figure 3-7. 3B7E_chainA_prepared set as the reference sequence.

In order to do some binding site analysis, we will first need to bring in the 3B7E sequence into the Multiple Sequence Viewer/Editor and then set it as a the reference so we can align the rest of the sequences to it

 

1. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, includethe entry is represented in the Workspace, the circle in the In column is blue 3B7E_chainA_prepared
2. In the Multiple Sequence Viewer/Editor panel, for Load from, choose Workspace and click the download icon
   • The sequence is added to the bottom of the MSV
3. Right-click on 3B7E_chainA_prepared
4. Click Set as Reference

Figure 3-8. Multiple sequence alignment with 3B7E_chainA_prepared as the reference.

5. Click Align
6. Unselect Selected only
7. Click Align
   • Sequences are re-aligned to 3B7E_chainA_prepared
8. Scroll to the right of the sequence viewer to look at the sequences starting with residue 84

Figure 3-9. Analyze Binding Site option in Tools.

9. Go to Select > Binding Site Residues
   • The binding site residues for the 3B7E_chainA_prepared structure are selected in the Multiple Sequence Viewer/Editor
10. Hover over the graph icon and click the …
11. Select Binding Site
    • The binding site region is annotated in the Multiple Sequence Viewer/Editor

Figure 3-10. Apply Multiple Sequence Viewer/Editor color to the Workspace.

We will now color the 3B7E_chainA_prepared structure such that the binding site residues are all red (based on the sequence).

 

12. Make sure the binding site residues are still selected, otherwise go to Select > Binding Site Residues to select them again.
13. Hover over the brush icon and click the …
14. Click Apply to and choose No residues on tab
15. For Paint selected, choose Red
16. Click Color entire residues
17. Click Color Residues

Figure 3-11. Binding site sequence coloring onto 3B7E_chainA_prepared.

18. Click the Presets dropdown
19. Choose Legacy > Pretty
    • The binding site residues are all colored red in the Workspace

Figure 4-1. Open Build Homology Model panel.

1. In the Multiple Sequence Viewer/Editor, right click on the second listed sequence gi|407943793|pdb|4B7Q|A and select Set as Reference
2. Click Other Tasks
3. Choose Build Homology Model
   • The Build Homology Model panel opens

Figure 4-2. Running a BLAST search to find homologs.

While we could use the 3B7E_chainA - prepared structure as a reference, we would like to simulate finding a homolog from a BLAST search

 

4. Make sure that Use is set to 1 target, 1 template
5. For Find a template structure, click Find
6. Click the cog
7. Uncheck Use local server only

 

Note: This requires internet access. The ‘Use local server only option’ is checked by default to prevent your BLAST searches and related tasks from going out to remote servers. To allow remote access, uncheck this option. You will be prompted to confirm the online search every time to avoid accidentally leaking information. This option is also available from the top-level Edit → Settings and Defaults menu.

 

8. Click Run Search

Figure 4-3. Sequence 2HTY_A selected in the BLAST Search results.

While there are many templates with 100% sequence identity which would surely be the most ideal if we were running this as part of a project, we will choose one with ~96% sequence identity, which though not perfect is still very good to better demonstrate the homology modeling process.

 

8. Choose ID 2HTY_H
9. Click Import
   • A new structure is includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspace the 3D display area in the center of the main window, where molecular structures are displayedand as an entry in the Multiple Sequence Viewer/Editor
   • The Find a template structure section in the Build Homology Model panel is now populated

Figure 4-4. Generate Model.

Note: With the high sequence identity between the two sequences we don’t need to run any alignment beyond the auto-alignment

 

Note: Make sure no ligands are selected in the Include ligands & cofactors (optional) section

 

10. For Job name, write homology_modeling_2HTY
11. Click Generate Model
    • This step takes ~1 minute
    • The structure is updated in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 4-5. The homology modeling results.

Note: Purple indicates regions that overlap directly with the template, cyan indicates regions where the target and template sequence differ

Figure 4-7. Structure Reliability Report in Biologics.

Now that we have predicted the structure of the protein via homology, we want to assess its quality compared to other structures in the Protein Data Bank. See the documentation page for Protein Reliability Report panel for more information.

 

1. Go to Biologics > Structure Reliability Report
   • The Protein Reliability Report opens

Figure 4-8. Run Protein Reliability Report job.

2. Change job name to homology_reliability
3. Click Run
   • This step takes ~2 minutes
   • A new entry is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
   • A banner appears

Figure 4-9. Protein Reliability Report results.

4. On the banner, click Protein Reliability Report
   • The Protein Reliability Report Panel Opens

Note: The Protein Reliability Report output can also be viewed by including homology_reliability-out in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and reopening the Protein Reliability Report Panel through Tasks > Biologics > Reliability Report

 

Figure 4-10. Clean Up stage of the Protein Preparation Workflow.

To address some of the issues found in the Protein Reliability Report we are going to do some structural refinement using the Protein Preparation Workflow.

5. Go to Tasks > Browse All > Protein Preparation and Refinement > Protein Preparation Workflow
   • The Protein Preparation Workflow panel opens
6. Click INTERACTIVE
7. Under Minimize and Delete Waters, go click Settings
8. Under Delete waters, uncheck Distant from ligands (hets)
9. Click Clean Up
   • This job takes ~2 minutes
   • A new entry is in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Figure 4-11. Results from the Protein Reliability Report of the prepared homology model.

10. In the Protein Reliability Report, change the job name to homology_reliability_prepared
11. Click Run
12. On the banner, click Protein Reliability Report
    • The Protein Reliability Report Panel Opens

Note: The biggest issues present in the Protein Reliability Report of the unprepared structure (steric clashes, and side chain and backbone dihedrals) have been fixed with the minimization.