Chimeric Homology Modeling Using the Multiple Sequence Viewer/Editor

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

Tutorial files

0.4 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 build a chimeric homology model of the beta-3 adrenergic receptor using beta-1 and beta-2 adrenergic receptors as templates. Beta-adrenergic receptors are a class of G protein-coupled receptors that are targets for epinephrine and norepinephrine.  Adrenergic receptors have been implicated in lipolysis and thermogenesis regulation in adipose tissue and play a prominent role in the modulation of cardiac function and angiogenesis. Beta-blockers are one well-known class of compounds that target adrenergic receptors by blocking the binding of epinephrine thus lowering blood pressure. Three isoforms of the beta adrenergic receptor have been identified: beta-1, beta-2 and beta-3. Structures have been determined of beta-1 and beta-2 adrenergic receptors, however the structure of beta-3 is still elusive.

 

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Aligning the Two Adrenergic Receptors

  1. Performing Multiple Sequence Alignment

  1. Building a Homology Model

  1. Assessing Protein Structure Quality

  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

 

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

 

Figure 1-2. Import Structure.

  1. Go to File > Import Structures
  2. Select adrenergic_prepared_structures.maegz
  3. Click Open

Figure 1-3. Save Project.

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

 

 

2. Aligning the Two Adrenergic Receptors

In this section we will align structures of beta-1 and beta-2 adrenergic receptors. Note that this step may be skipped in favor of performing the structural alignment in section 4 when the homology model is built. However, a structure alignment is required if multiple templates are used to generate the homology model.

Figure 2-1. Open the Protein Structure Alignment panel.

  1. Go to Tasks > Browse > Structure Alignment > Protein Structure Alignment

 

Note: Alternatively you can just search Protein Structure Alignment

Figure 2-2. Include beta-1 and beta-2 adrenergic receptors structures.

  1. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, shift-click to includethe entry is represented in the Workspace, the circle in the In column is blue 2VT4_prepared_beta1 and 2RH1_prepared_beta2

 

 

2RH1_prepared_beta includes the sequence information of the fusion protein included in place of one of the loops (ICL3) to facilitate protein crystallization.

Figure 2-3. Align structures of beta-1 and beta-2 adrenergic receptors.

In other situations you may be interested in aligning just by the protein backbone, or by a certain collection of residues (by ASL). See the Atom Specification Language documentation for more information on ASL.

 

  1. In the Protein Structure Alignment panel, choose Workspace (included entries)
  2. Click Align
    • The Protein Structure Alignment Results panel opens
    • A smaller the alignment score indicates better overall alignment

 

 

These adrenergic structures have been prepared for you to include a single copy of the receptor and any artifacts of crystallization have been deleted. For more information about preparation of protein structures, see the Introduction to Structure Visualization and Preparation. Also see the Best Practices for Protein Preparation for more information.

Figure 2-4. Protein Structure Alignment results.

  1. In Protein Structure Alignment Results, click Close
  2. Close Protein Structure Alignment

An RMSD of ~1.2 Angstroms is more than sufficient for us to consider this a high quality alignment. You may want to try some visual inspection if there is any particular region that you would want to ensure is aligned properly.

3. Performing Multiple Sequence Alignment

In this section we will perform a multiple sequence alignment of the human beta-1, beta-2 and beta-3 adrenergic receptors sequences obtained from UniProt and the structures used in section 2.  The number and choice of templates and their sequence alignment will have a significant effect on the quality of the homology model built. The approach to build an optimal model may differ depending on the type of structure investigated. It is recommended to use multiple high similarity sequences if possible. However, if multiple sequences are available, but only one has high similarity, it may be better to forgo the extra sequences.

Figure 3-1. Open the Multiple Sequence Viewer/Editor.

  1. Go to Tasks > Biologics > Multiple Sequence Viewer/Editor
    • The Multiple Sequence Viewer/Editor opens
    • The sequences 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 sequences are shown

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

  1. In the MSV, go to  File > Import Sequences from File

Figure 3-4. Importing sequences.

Load a file that contains human sequences of beta-1, beta-2, and beta-3 adrenergic receptors obtained from UniProt

 

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

 

 

Figure 3-5. Select the reference sequence.

  1. Click then right-click on BETA-3_H
  2. Choose Set as Reference

Figure 3-6. Align sequences.

  1. Click Align
  2. For Using, choose Multiple Sequence Alignment
  3. Make sure Selected only is unselected
  4. Click Align

 

 

 

4. Building a Homology Model

In the section, we will build a homology model of the beta-3 adrenergic receptor using beta-2 adrenergic receptor as the template for TM3 and TM4 and beta-1 adrenergic receptor structure as a template for the rest of the structure.

Figure 4-1. Open Build Homology Model panel.

  1. In the MSV, go to Other Tasks > Build Homology Model
    • The Build Homology Model panel opens

Figure 4-2. Choose setting.

  1. For Use, choose Multiple templates (chimera)

 

Figure 4-3. Select template regions.

Drag the sequences to make sure that 2VT4_prepared_beta1 is above 2RH1_prepared_beta2 in the Multiple Sequence Viewer/Editor. Also make sure that both are selected

 

  1. For Define regions on templates, click Pick
    • The top template sequence (2VT4_prepared_beta1) is colored yellow
  2. Select column index 131-197 in the 2RH1_prepared_beta2 row until they are all colored yellow

 

Note: The yellow specifies which regions to take from each structure template to create the chimeric model

Figure 4-4. Add ligands to the final model.

  1. For Include ligands and cofactors (optional), click Choose
  2. Select just A: CAU 408
  3. Click OK

 

 

 

 

 

Figure 4-5. Generate homology model.

  1. For Change model settings (optional), click View Settings
  2. For Method, choose Energy Based
    • This will take more time than the Knowledge-based method, but will make a much better model
  3. Click OK
  4. For Job name, type homology_modeling_chimera
  5. Click Generate Model
    • This job will take 10-15 minutes to complete
  6. Include the entry titled Chimeric Model of Beta-3_H based on 2VT4_prepared_beta1_C, 2RH1_prepared_beta2_A-1 in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 4-6. Chimeric model of the beta-3 adrenergic receptor.

Note: The output structure is 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 side chain 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

5. Assessing Protein Structure Quality

Figure 5-1. Reliability Report in Biologics Tasks.

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 5-2. Protein Reliability Report.

  1. In Analyze, choose Entire structure
  2. Change the Job name to chimeric_model
  3. Click Run
    • This job takes about a minute to complete
    • 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 5-3. Reliability Report output.

  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 chimeric_model-reliability-out
    • The Protein Reliability Report is visualized

 

 

The size of the circle relates to the magnitude of the metric. A circle is red if the deviation from acceptable values is larger than a threshold, and green if the deviation is smaller. White is neutral or no information.

 

If the missing loop proves to be an issue for your modeling tasks, you can use the Refine Loops panel to build in and refine the loop.

6. Conclusion and References

In this tutorial, we used two different structure templates to build a chimeric model.  Structures of beta-1 and beta-2 adrenergic receptors were used to build a chimeric model of the beta-3 adrenergic receptor.

For further learning:

 

7. 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

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