Building Homology Models with the Multiple Sequence Viewer/Editor

Tutorial Created with Software Release: 2024-2
Topics: Antibody Design, Biologics Drug Discovery, Enzyme Engineering, Structure Prediction & Target Enablement
Products Used: BioLuminate

Tutorial files

8.6 MB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayedthe 3D display area in the center of the main window, where molecular structures are displayed

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

In this tutorial, you will learn how to build a homology model of the human protein factor Xa using sequence information as a template.

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Building a Model and Assessing Quality

  1. Refining a Homology Model

  1. Conclusion and References

  1. Glossary of Terms

1. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location that files are saved in BioLuminate to make file navigation easier. Each session in BioLuminate begins with a default Scratch Projecta temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project, which is not saved. A BioLuminate project stores all your data and has a .prj extension. A project may contain numerous entries corresponding to imported structures, as well as the output of modeling-related tasks. Once a project is created, the project is automatically saved each time a change is made.

Structures can be imported from the PDB directly, or from your Working Directorythe location that files are saved using File > Import Structures, and are added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data. The Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion is located to the left of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. The Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data can be accessed by Ctrl+T (Cmd+T) or Window > Project Table if you would like to see an expanded view of your project data. The Toggle Table is another way to interact with structures and can be accessed via Window > Toggle Table.

  1. Double-click the BioLuminate icon

All of the workflows shown here can also be done in Maestro

Figure 1-1. Change Working Directory option.

  1. Go to File > Change Working Directory
  2. Find your directory, and click Choose
  3. 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/hm_msv.zip
  4. After downloading the zip file, unzip the contents in your Working Directory for ease of access throughout the tutorial

 

Figure 1-2. Open Project.

  1. Go to File > Open Project
  2. Choose FXa_homologymodeling.prjzip
  3. Click Open
    • Structures are shown in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
    • A structure is 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
  1. In the Save scratch project warning box, click OK

 

Figure 1-3. Save Project.

  1. Go to File > Save Project As
  2. Change the File name to MSV_homology
  3. Click Save
    • The project is now named MSV_homology.prj  

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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

2. Building a Model and Assessing Quality

Sometimes structure-based drug design projects lack a suitable 3D structure of the protein of interest and only the primary sequence information is available. Homology modeling can be used to build a 3D model using a suitable homolog, or homologs, which act as a structural template for the sequence. Homology modeling works on the premise that sequence similarity can equate to structural similarity.

2.1     Build a homology model

Figure 2-1. Multiple Sequence Viewer/Editor option in Biologics Tasks.

  1. Go to Tasks > Biologics > Multiple Sequence Viewer/Editor
    • The Multiple Sequence Viewer/Editor panel opens

Figure 2-2. The Multiple Sequence Viewer/Editor panel.

  1. Go to File > Import Sequences from File
  2. Choose factorXa_human.fasta and click Open
  3. Click Other Task and choose Build Homology Model
    • The Build Homology panel opens

Figure 2-3. Find Homologs.

  1. For Find a Template Structure, click Find
    • A dialog appears
  2. Click the cog
  3. 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 (after a confirmation), clear this option. It is also available from the top-level Edit → Settings and Defaults menu.

 

  1. Click Run Search

Figure 2-4. BLAST Search Results panel with 1K1G_H chosen.

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 ~84% sequence identity, which though not perfect is still very good to better demonstrate the homology modeling process.

 

 

  1. In the BLAST Search Results panel, choose 1KIG_H
  2. Click Import

Figure 2-5. Run the Homology Model job.

Note: The auto-align of the sequences when the template was imported was sufficient, so we do not need to proceed with any further alignment

 

  1. For Job name write homology_modeling_FXa
  2. Click Generate Model
    • The job should take a few minutes
    • Once completed, 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 homology model annotated by color.

Note: Output structure annotated by color

  • Blue - full residue conformation is copied from the template, identical residues are at this position
  • Cyan - residue backbone conformation is copied from the template, a sidechain mutation is at this position
  • Red - residue backbone and sidechain positions have been modeled, an insertion, deletion, or experimental constraint requires this position to be predicted

2.2     Assess model quality

Figure 2-7. 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 Tasks > Biologics > Structure Reliability Report
    • The Protein Reliability Report opens

Figure 2-8. Protein Reliability Report panel.

  1. Change job name to homology_reliability
  2. 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 2-9. The Protein Reliability Report results.

  1. 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

Note: Click on any of the circles for a more in-depth analysis

 

Note: In the Protein Reliability Report, small green circles represent properties within a reasonable distance of the average PDB structure, while large red circles are considered potential matters of concern. In general, the three outputs that are looked at to determine the quality of a protein structure are ‘Steric Clashes’, ‘Missing Atoms’ and ‘Missing Loops’

3. Refining a Homology Model

After building a model, you can examine its quality and fix problems, such as steric clashes, prior to using the model for further calculations. Two tools for refining structures are the Protein Preparation Workflow and Prime Loop Refinement. Most common structural problems can be resolved using the Protein Preparation Workflow, while Prime Loop Refinement can address more complex issues. Prime Loop Refinement is a time and CPU-intensive calculation, so the Protein Preparation Workflow is the recommended starting point for model refinement. Please see the Introduction to Structure Preparation and Visualization tutorial for more details on using the Protein Preparation Workflow. Also see the Best Practices for Protein Preparation for more information.

Figure 3-1. The Refine tab of the Protein Preparation Wizard.

  1. In the Favorites Bar, Click Protein Preparation
    • The Protein Preparation Workflow panel opens
  2. Click INTERACTIVE
  3. Under Optimize H-bond Assignments, click Optimize
    • This step takes about a minute
    • 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
    • The overlapping atoms have been corrected, and side chains that have been flipped are now labeled in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
  4. Under Minimize and Delete Water, click Clean Up
    • This step takes about a minute
    • 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

Note: Hover over buttons to find out more about their function

Figure 3-2. Examine Protein Reliability Report results.

  1. In the Protein Reliability Report, change job name homology_reliability_prepared
  2. 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
  3. On the banner, click Protein Reliability Report
    • The Protein Reliability Report Panel Opens

Note: There are large improvements in Steric Clashes and Bond Angle Deviations compared to the previous results.

4. Conclusion and References

This short tutorial explored the building homology models using the Multiple Sequence Viewer/Editor.  We built a homology model of the human Factor Xa using 1KIG, a bovine ortholog, as the template. We assessed the quality of the model by generating a Structure Reliability Report. Finally, we used the Protein Preparation Workflow to refine the structure and fix problems that were associated with the model.

For further learning:

 

5. Glossary of Terms

BLAST - Basic Local Alignment Search Tool, used for calculating sequence similarity

Entry List - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

included - the entry is represented in the Workspace, the circle in the In column is blue

incorporated - once a job is finished, output files from the working directory are added to the project and shown in the Entry List and Project Table

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project

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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location that files are saved

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed