Sequence Annotation of Antibodies with the Multiple Sequence Viewer/Editor

Tutorial Created with Software Release: 2024-2
Topics: Antibody Design, Biologics Drug Discovery
Products Used: BioLuminate

Tutorial files

13 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 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 use custom sequence annotations in the Multiple Sequence Viewer/Editor to map residue-level aggregation properties onto the sequence.

 

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Analyzing the Antibody Surface

  1. Annotating the Antibody Sequence in the Multiple Sequence Viewer/Editor

  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 Maestro to make file navigation easier. Each session in Maestro 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 Maestro 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.

  1. Double-click the BioLuminate icon

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/antibody_annotation.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 Sequence_Annotation.prjzip
  3. 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 1-3. Save Project panel.

  1. Go to File > Save Project As
  2. Change the File name to sequence_annotation , click Save
    • The project is now named sequence_annotation.prj

2. Analyzing the Antibody Surface

In this section, we will generate an aggregation surface for the 4M5Z structure. This is useful for predicting residue patches that may encourage aggregation in solution.

Figure 2-1. Protein Surface Analyzer in Biologics.

Here we will select just the antibody structure in the workspace (from the antibody-antigen complex) so we can load just that into the Protein Surface Analyzer. For more information on protein surface analysis check out the Liability Analysis for Biologics tutorial or the Protein Surface Analyzer documentation page

 

  1. Include 4M5Z in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
  2. Type C on your keyboard
    • Selection mode is changed to Chains
    • The cursor has a ‘C’ next to the arrow
  3. 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 both Chain H (blue) and Chain L (red)
  4. Go to Tasks > Biologics > Protein Surface Analyzer

 

Note: Use the Structure Hierarchy or Status Bar to help identify the chains

Figure 2-2. Protein Surface Analyzer panel.

  1. For Use structure from, choose Workspace (selected residues only)
  2. Click Analyze
    • This job takes about a minute
    • An ‘S’ icon is in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Note: Previous analyses can be imported from file to the Protein Surface Analyzer panel. Additionally, completed analyses can be saved for later reference.

Figure 2-3. Patch Browser tab of the Protein Surface Analyzer.

  1. Check Fit to select
  2. Click on a Patch entry
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is zoomed to the selection

 

Note: Click Settings to change colors, size cutoff, value cutoff, etc.

Figure 2-4. Aggregation table sorted by AggScore.

  1. Click the Aggregation tab
  2. Click the AggScore header twice
    • The table is now sorted by Aggscore
  3. For Color by, choose AggScore
    • The table and surface patches are now colored by AggScore

 

 

Note: AggScore provides a single numerical value to estimate the aggregation propensity of a protein based on a patch analysis of the protein surface. The score is based on the respective contribution of residues to hydrophobic and charged surface patches smoothed over a residue window of five consecutive residues. The method is strictly structure-based, not dependent on the presence of natural amino acids and is not trained on beta-amyloid aggregation data. The AggScore function provides propensity values for each amino acid position in a protein, thus allowing for the prediction of aggregation hotspots within the protein.

Figure 2-5. Aggregation Profiles panel.

We will now  look at the per-residue contribution by chain (according to AggScore as well as Aggrescan and Zyggregator)

 

Click Profiles

  • Aggregation profiles for chains are shown

 

 

3. Annotating the Antibody Sequence in the Multiple Sequence Viewer/Editor

In this section, we will use custom sequence annotation to color the residues by their Aggscore values.

3.1     Color sequence in Multiple Sequence Viewer/Editor by AggScore

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

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

Figure 3-2. Split chains.

  1. Click the + icon in the bottom right-hand corner of the Multiple Sequence Viewer/Editor and make sure Split Chains is selected
    • All chains in this entry are split in the Multiple Sequence Viewer/Editor

Figure 3-3. Edit sequence coloring.

 

  1. Hover over the brush icon and click the …
  2. For Color by, choose Define Custom Scheme

Figure 3-4. Define Custom Scheme by AggScore.

  1. For Start with, choose Residue Property
  2. For Residue Property, choose residue aggscore
  3. For Color palette, choose Blue-White-Red
  4. For # values/ranges, type 10
  5. Click Update
  6. Click Save
    • The updated coloring has been added to the Multiple Sequence Viewer/Editor

Note: Any residue-level property can be mapped onto the sequence in a similar manner

3.2     Annotate the CDRs

Figure 3-5. Add Antibody CDR annotation.

  1. Hover over the chart icon and click the …
  2. Click Antibody CDRs
    • The antibody CDR regions are now annotated in the Multiple Sequence Viewer/Editor

Note: After selecting Antibody CDRs, you can choose which numbering scheme you would like to use for the annotation and whether to annotate the Vernier Zone

Figure 3-6. Change the numbering scheme.

  1. Change the numbering scheme to Chothia
    • The annotation is updated in the Multiple Sequence Viewer/Editor

4. Conclusion and References

In this tutorial, we generated residue-level aggregation properties using the Protein Surface Analyzer, and then mapped those customs properties onto the sequence as a color gradient.

5. Glossary of Terms

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

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