Heteromultimer Homology Modeling with 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 heteromultimer homology model of human hemoglobin from a bar-head goose hemoglobin structure.

 

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Building and Evaluating a Heteromultimer 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 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 Maestro 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/heteromultimer_msv.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location that files are saved for ease of access throughout the tutorial  

Figure 1-2. Open Project panel.

  1. Go to File > Open Project and choose heteromultimer.prjzip
  2. 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
  3. In Save scratch project, click OK
  4. Go to File > Save Project As
  5. Change the File name to heteromultimer_msv click Save
    • The project is named heteromultimer_msv.prj
    • 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

2. Building and Evaluating a Heteromultimer Homology Model

Hemoglobin is a four-chained oxygen-transport metalloprotein (typically) composed of an alpha and beta subunit. While the sequence identity for both the alpha and beta subunits among primates is nearly 100%, as you get to more distantly related species the sequence identity drops.

While there have been high quality (< 2.5 Å) crystal structures of human hemoglobin since the late 1970s, we are going to simulate what a researcher might do if they did not have access to a human hemoglobin structure, but did have access to a bar-head goose structure (PDB ID: 1HV4). Since we would like to predict the structure of both the alpha and beta subunits together, we will need to build a heteromultimer model using the Multiple Sequence Viewer/Editor. After building the model, we will compare the predicted structure to that of a human hemoglobin crystal structure to evaluate the quality of the prediction.

2.1     Build a heteromultimer 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. Import Sequences.

  1. Go to File > Import Sequences from File
  2. Choose hemo_human.fasta and click Open
    • A new tab is added called Workspace Copy, which includes all 4 chains of the human hemoglobin sequence

 

Figure 2-3. Create and rearrange tabs.

For the heteromultimer workflow, we will need to have separate Views in the Mulitiple Sequence Viewer/Editor, each one with a matching pair for reference structure and sequence.

 

  1. Click the + icon next to the Workspace Copy tab to create View 3, View 4, and View 5
  2. Rearrange the tabs so the Workspace Copy tab is 2nd in to the left, and View 1 is followed by View 3,View 4 and View 5

 

Figure 2-4. Duplicate query sequences into new tabs.

  1. Go to the Workspace Copy tab
  2. Right-click the Hemoglobin_Human_A sequence
  3. Go to Duplicate > Into Existing Tab > View 1
  4. Repeat steps 7 and 8 for to duplicate chain B to View 3, chain C to View 4, and chain D to View 5

Figure 2-5. Duplicate template sequences into new tabs.

  1. In BioLuminate, include the Bar-Head Goose Hemoglobin (1HV4) entry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
  2. In the Multiple Sequence Viewer/Editor, go to the Workspace tab
  3. Right-click the Bar-Head Goose Hemoglobin Chain A sequence
  4. Go to Duplicate > Into Existing Tab > View 1
  5. Repeat steps 12 and 13 for to duplicate chain B to View 3, chain C to View 4, and chain D to View 5

Figure 2-6. Open the Build Homology panel in the Multiple Sequence Viewer/Editor.

  1. Switch to the View 1 tab
  2. Click Other Task and choose Build Homology Model
    • The Build Homology panel opens

Figure 2-7. Choose the Multiple View tabs (heteromultimer) option in the Build Homology Model panel.

  1. For Use, choose Multiple View tab (heteromultimer)
  2. Click the Views 1, 3, 4 and 5 to select them all

Note: The Workspace Copy row should be the only one not highlighted in blue.

Figure 2-8. Set up the homology model job.

  1. Click the cog icon next to View 1
    • The Set Up tab opens

Note: Since we already copied our template sequences to the View 1, View 3, View 4, and View tabs the templates will be automatically recognized in the Set Up tab of the Build Homology Model panel.

Note: The sequences are already aligned so you do not need to click Run Alignment.

  1. Click Choose next to Include ligands & cofactors, and make sure HEM (the heme) is selected
  2. Click Create Tabs
  3. Repeat steps 23 and 24 for View 3, View 4, and View 5

Figure 2-9. Run Homology Modeling job.

  1. Next to Job name, type homology_modeling_hemo_human
  2. Click Generate Model
    • This job should take around 2-3 minutes
    • Once the job is completed the model with be incorporated in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

2.2     Align the predicted human hemoglobin structure with a crystal structure

Figure 2-10. View Homology Model output in the Workspace.

To evaluate the quality of our homology model, we will align it to a relatively more recent crystal structure of human hemoglobin to see how we would have done with the information available to us at the time.

  1. Include the Model of Hemoglobin_Human… entry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

 

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

Figure 2-11. Download the 2HBB crystal structure from the PDB.

  1. Go to File > Get PDB
  2. Next to PDB IDs, type 2HHB

Note: This is a high-quality crystal structure of human hemoglobin. See PDB entry here.

  1. Click Download
    • The 2HHB structure has been added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and included in he Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Note: Both the Model of Hemoglobin_Human… and 2HHB entries should be included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 2-12. Workspace tab in the Multiple Sequence Viewer/Editor.

  1. Go to the Workspace tab in the Multiple Sequence Viewer/Editor
  2. Click the + icon in the bottom right-hand corner and unselect Split Chains

Figure 2-13. Align the homology model with the crystal structure.

  1. Go to Align, and select Structures
  2. Next to Using, choose Protein Structure Alignment
  3. Click the cog and select Entire Entry
    • Each sequence will represent the entire entry

Note: Make sure Selected Only is toggled off

  1. Click Align

Note: When aligning structures, the top structure will always be viewed as the reference.

Figure 2-14. Protein Structure Alignment Results.

Note: Once the alignment is complete, a popup appears showing that the RMSD is ~2.5 Angstroms

  1. Click OK
    • The popup is closed

Figure 2-15. Use the BioLuminate Default Preset to better visualize the alignment of the two structures.

  1. In BioLuminate, go to Presets > BioLuminate Default
    • You can now better visualize the quality of the alignment  

3. Conclusion and References

In this tutorial, we generated a homology model of human hemoglobin from a bar-head goose hemoglobin structure, and then aligned the generated model with a human hemoglobin crystal structure to evaluate the model quality.

4. 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