Analyzing Binding Sites of Nucleic Acids with SiteMap

Tutorial Created with Software Release: 2025-3
Topics: Small Molecule Drug Discovery, Structure Prediction & Target Enablement
Products Used: Maestro, SiteMap

Tutorial files

14.6 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 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:

 

This tutorial will guide you through essential steps of identifying and evaluating potential binding sites in nucleic acid structures using SiteMap.

 

Tutorial Content

  1. Introduction to SiteMap

  1. Creating Projects and Importing Structures

  1. Analyzing and Evaluating Binding Sites with SiteMap

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to SiteMap

Structure-based drug discovery approaches typically require the knowledge of the target structure bound to a ligand. SiteMap is a tool for identifying and evaluating the binding sites present in the target structure. The identified sites can be mapped, scored, and visualized to understand how well the ligand fits in the binding pocket. This also provides insights for modifying ligands for improved binding affinity and overall stability of ligand-receptor complex.

Unlike proteins, RNAs are quite flexible and can fold into multiple stable conformations. Binding of small ligand molecules often induces structural and conformational changes in an RNA receptor. Further, ligand binding sites on RNAs tend to be shallow, more polar, and conformationally flexible.

SiteMap is an effective tool for analyzing DNA/RNA binding sites, alongside its ability to identify and evaluate protein binding sites. A SiteMap calculation consists of three stages. In the search stage, sites are identified via groups of site points on a grid, which must be located outside but near the surface of the receptor and must be sufficiently enclosed. In the subsequent mapping stage, contour maps are generated based on interactions with the receptor. The final evaluation stage calculates various properties to determine whether a site is likely to be able to accommodate a drug-like binder favourably.

This tutorial uses a prepared 6DN3 RNA structure, which is a flavin mononucleotide (FMN) riboswitch bound to a flavin analog ligand BRX1555. If you are following along with a different structure, please make sure that your structure is prepared as shown in the Preparing Nucleic Acid Structures tutorial.

In this tutorial, you will learn how to analyze the potential binding sites in the nucleic acid structures using the SiteMap panel. For detailed information, please refer to the SiteMap panel documentation and Target Analysis with SiteMap and WaterMap tutorial.

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location where 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 saved, the project is automatically saved each time a change is made.

Structures can be built in Maestro or can be imported using File > Import Structures (or drag-and-dropped), and are added to the Entriesa 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 Entriesa 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 2-1. Change Working Directory option.

  1. Go to File > Change Working Directory.
  2. Find your directory, and click Choose.
  3. Pre-generated 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/sitemap_nucleic_acid.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

Figure 2-2. Saving the project.

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

Figure 2-3. Importing the prepared structure.

  1. Go to File > Import Structures.
  2. Navigate to and find RNAprep_6DN3-out.maegz.
  3. Click Open.
    • A new entry titled 6DN3 - prepared is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and a banner appears confirming successful import of the structure.

Note: Imported structures in Maestro are 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 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion by default. Please refer to the Glossary of Terms for the difference 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

3. Analyzing and Evaluating Binding Sites with SiteMap

In this section, you will analyze the potential binding sites in the 6DN3 RNA structure using the SiteMap panel. You will also quantitatively evaluate how likely each pocket is a good binding site by comparing the SiteScore for each pocket.

Figure 3-1. Splitting the prepared structure.

In order to find, map and evaluate potential binding sites, SiteMap requires a prepared structure without any ligands, solvents, or other molecules. Everything present in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed will be considered as part of the receptor.

 

  1. Right-click on 6DN3 - prepared and choose Split > Into Ligands, Water, Other.
    • The original entry is split into individual entries.

Figure 3-2. Renaming and including the apo structure in the Workspace.

  1. Double-click the 6DN3 - prepared_protein and rename it to 6DN3 - prepared_RNA_apo.
  2. Includethe entry is represented in the Workspace, the circle in the In column is blue 6DN3 - prepared_RNA_apo.

Figure 3-3. The Binding Site Detection in Tasks.

  1. Go to Tasks > Browse > Structure Analysis > Binding Site Detection.
    • The SiteMap panel opens.

 

SiteMap allows you to examine the entire receptor for potential binding sites through the “Identify top-ranked potential receptor binding sites” option, or restrict the search to a given region through the “Evaluate single binding site region” option.

 

Running a SiteMap calculation in Identify mode:

  • If your structure has multiple binding pockets, this mode can identify, rank and give insights into their physicochemical properties such as size, enclosure, hydrophilicity and hydrogen bond donor/acceptor character of each pocket.
  • If your structure has a known ligand binding site, this mode can validate and rank your known binding site among other potential sites.
  • If you are working with an apo structure of the target, this mode can help you define a receptor grid for performing docking calculations.

 

Running a SiteMap calculation in Evaluate mode:

  • If you only need to analyze a specific region around a ligand, this mode is helpful.
  • If you have identified a potential binding pocket through obtaining a ligand-bound experimental structure, mixed-solvent molecular dynamics or pharmacophore modeling, then this mode is helpful to evaluate the characteristics and druggability of that pocket.

Figure 3-4. Running the job with the default settings.

In this example, you will use the Identify mode to gain insights into all potential pockets on the riboswitch’s surface.

 

  1. For Task, choose Identify top-ranked potential receptor binding sites.
  2. Change the Job name to sitemap_6DN3_apo.
  3. Click Run to start the job.
    • The job takes ~1 minute.
    • A banner appears and a new group sitemap_6DN3_apo_out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  4. Close the SiteMap panel.

Figure 3-5. The Show Property option.

The SiteMap results have two components: the site representation in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, and the various scoring metrics saved as entry properties.

 

  1. In the top-right corner of Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, click the Change table settings icon (three vertical dots) and choose Show Property.
    • The Show Properties in table dialog box opens.

Figure 3-6. Adding SiteScore and Dscore to compare the identified sites.

  1. In the dialog box, click Choose.
  2. From the list, find and select SiteScore and Dscore.
  3. Click OK.
    • Columns for the selected properties are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.  

Optional: You can also search for and add volume and balance. The volume gives an idea of the ability of a site to accommodate a ligand of a certain size. The balance is a measure of whether a site contains a good mixture of hydrophilic and hydrophobic regions for a ligand to bind to.

The SiteScore incorporates the number of site points, the degree of enclosure, and the hydrophilicity of sites. It generally distinguishes between binding and non-binding sites, with higher SiteScores indicating a higher propensity for molecule binding. On the other hand, the Dscore assesses the druggability of a binding site i.e. the likelihood that the site can effectively bind small drug-like molecules with high affinity. The Dscore also incorporates the same properties as SiteScore, but with a critical difference. Unlike the SiteScore, the Dscore does not limit the hydrophilic term. This means it more heavily penalizes highly polar or charged areas, which are less likely to bind typical drug-like compounds. To know more, please refer to the SiteMap methodology documentation.

 

SiteScores and Dscores above 1.0 are considered good, as they are calculated relative to the average of a large number of tight-binders. Note that a site can bind molecules tightly (have a good SiteScore), but still be non-druggable (have a bad DScore), because drug-like molecules are unlikely to bind.

 

SiteScore was recently rebalanced to improve the detection of RNA binding sites. The details of this optimization can be found in this publication.

Figure 3-7. Setting up the Workspace for visualizing the SiteMap results.

 

  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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries sitemap_6DN3_apo_out group by clicking on the group heading.
  2. Double-click the In circle of 6DN3 - prepared_ligand and sitemap_6DN3_apo_protein entries to to fix both the entries in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: The entry sitemap_6DN3_apo_protein contains the RNA receptor irrespective of the name ending with “protein”.

 

Optional: Choose a visualization that is most helpful to you. For example, in this case, the ligand is displayed in the ball-and-stick representation, the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is zoomed to the ligand, and only the ligand-receptor interactions are shown for clarity.

Figure 3-8. Visualizing the SiteMap results.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue the sites found by SiteMap one at a time and use the right and left arrow keys to visually analyze them in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Take into account the corresponding properties in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.

The generated Site Map shows the site points (white spheres), hydrophobic (yellow), hydrogen-bond donor (blue) and acceptor (red) maps. The top-ranked site is the active site, to which the cocrystallized ligand BRX1555 binds. It has acceptable SiteScore and Dscore values. The found site points and different maps align well with the ligand. The hydrophobic groups on the ligand can be seen occupying hydrophobic regions, and the donors and acceptors of the ligand are located in or very close to the appropriate donor and acceptor regions.

Figure 3-9. Hiding the white spheres for clarity.

Optional: You can hide the white spheres to declutter the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed by toggling off the Other checkbox in the Hierarchy.

4. Conclusion and References

In this tutorial, you learned how to identify potential binding sites in a nucleic acid structure. You also learned to quantitatively evaluate how likely each pocket is to be a good binding site by comparing the SiteScore for each pocket.

You can now for example use the insights into the receptor structure and ligand interactions for building a docking model, or optimizing the ligand further using the Ligand Designer. See the further learning section below or the Oligonucleotide Modeling learning path for additional resources.

5. Glossary of Terms

Entries - 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

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

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

Working Directory - the location where files are saved

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