Structure Visualization and Interaction Analysis in Nucleic Acids

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

Tutorial files

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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 visualizing the key interactions present within the nucleic acid structures.

 

Tutorial Content

  1. Introduction to Structure Visualization

  1. Creating Projects and Importing Structures

  1. Visualizing the Structure

  1. Creating a Custom Set

  1. Analyzing the Interactions

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Structure Visualization

Biomolecules are highly complex with unique three dimensional structures and diverse functions. Structure visualization plays an important role in the study of biomolecules. This helps one to understand components of the structure, spatial arrangements of atoms and connectivity of chemical bonds. It gives valuable insights about how the molecular properties are related to the structure. Visualization techniques can address some simple yet important questions:

  • How flexible is the target?
  • Does the target have a ligand bound to it?
  • Which residues are playing a crucial role in the binding site if the target has a bound ligand?
  • What are the various interactions present between the ligand and the receptor?

Maestro provides an easy-to-use interface for visualizing molecules, from small compounds to large biomolecules like proteins, nucleic acids, etc. It offers a wide range of display styles to represent molecules and customize color schemes based on various properties.

This tutorial uses the 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 apply visual representations to your structure. You will also learn how to create a custom set, and visually analyze key interactions within the ligand-receptor system in 2D and 3D.

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/visualization_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. The Save Project panel.

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

In this section, you will visualize key interactions within the 6DN3 FMN riboswitch structure, identifying the canonical and non-canonical base pairs. You will also visualize the interactions between the RNA receptor and flavin-analog ligand in the 2D Ligand Interaction Diagram. For an in-depth understanding of styling features, please refer to the Introduction to Structure Preparation and Visualization tutorial.

3.1 Visualize the structure

Figure 3-1. Selecting the nucleic acid (RNA) structure.

Different representation models can be used to illustrate the 3D-configuration and spatial arrangement of atoms in biomolecules. Visual representations like ribbon diagrams can help understand how biomolecules fold into specific secondary structure elements.

 

  1. Under Quick Select, click the choose item button (three horizontal dots) and choose Nucleic Acids.
    • All the atoms, excluding the ligand, are selected in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 3-2. Applying Ribbons to the RNA structure using the Style Toolbox.

Each object’s representation can be changed using the Style Toolbox.

 

  1. Go to the Style toolbox and click Ribbons.
    • The structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed appears in cartoon styled ribbons.
    • The Edit Ribbon menu appears.
  2. For Color scheme, choose Chain.
  3. Click the Fit-all button to view the entire structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • Take your time to visualize the secondary and tertiary structural elements.

The 6DN3 structure is a split RNA, representing a segment of larger functional molecule. It consists of two chains, X and Y, colored in red and orange respectively.

Figure 3-3. Applying ball-and-stick representation to the ligand using the Style Toolbox.

  1. Under Quick Select, click L.
    • All the ligand atoms are selected in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Go to the Style Toolbox and choose ball-and-stick representation.
  3. Click Fit view to ligand button.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to the ligand.
    • Take your time to visualize the ligand structure.

Figure 3-4. Applying Default Custom Preset to the structure.

Presets are available to quickly apply common styles to biomolecules to facilitate easy visualization. These can be used in a variety of ways, from decluttering your structure to creating publication-quality images.

 

  1. Double-click Presets.
    • The default Custom Preset is applied to the structure and the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to the ligand.
  2. Click the Fit-all button to view the entire structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: The default custom preset can be modified via the Edit Default Custom Preset option in the Presets menu.

The 6DN3 structure is a multi-stem junctional riboswitch centered on a flavin analog ligand BRX1555 (GZ4 201) bound to the junctional region. The junctional region is stapled together by two domains (orange and blue colored regions forming one domain; green and yellow colored regions forming the other) resulting in an overall butterfly-like fold. Each domain consists of two interacting stem-loop regions, the loops being formed by the following residues: X:17 - X:25 (Loop 1), X:36 - X:43 (Loop 2), Y:68 - Y:76 (Loop 3), Y:88 - Y:95 (Loop 4).

4. Creating a Custom Set

Custom Sets in Maestro allow you to manage and work with atom/residue selections within your project. These are particularly helpful for complex selections you frequently need. In this section, you will create a Custom Set for the side chains in 6DN3 RNA structure.

Figure 4-1. The Manage Sets option in Quick Select.

  1. Under Quick Select, click the choose item button (three horizontal dots).
  2. In the Custom Sets section, choose Manage Sets.
    • The Manage Sets dialog box opens.
  3. In the dialog box, click Add.
    • The Create Custom Set dialog box opens.

 

 

Figure 4-2. Creating a Custom Set for the side chains in 6DN3 structure.

  1. From the list on the left, choose Nucleic Acids (all or subset).
  2. For the subset, choose RNA and Side chain only.
  3. Change the Set Name to 6DN3_RNA_side_chain.
  4. Click Save Set.
    • A new Custom Set is created which can be accessed from the Custom Sets section in Quick Select.
  5. Close the Manage Sets dialog box.

 

Note: You can further refine the selection with the Add condition, Also Select, and Step buttons. This will allow you to make complex selections based on various criteria such as residue types, distances, atom properties, etc. You can also define a selection set from a workspace selection. For more information, see Selecting Atoms.

Figure 4-3. Selecting the side chain atoms via the created Custom Set.

You can now use the created custom set to quickly change the style of all RNA side chains.

 

  1. Under Quick Select, click the choose item button.
  2. In the Custom Sets section, select 6DN3_RNA_side_chain.
    • All the side chain atoms are selected in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-4. Applying thin tube representation to the side chain atoms.

  1. Go to the Style Toolbox and choose the thin tube representation.

5. Analyzing the Interactions

Various interactions, including hydrogen bonds, electrostatic interactions, pi interactions, and van der Waals forces, etc. play a crucial role in structural stability and function of biomolecules. These interactions govern how the biomolecules fold, adopt unique three dimensional structure, and how the ligands recognize and bind to the targets. By analyzing these interactions, you can identify key residues involved in ligand binding, explore structure-activity relationships, and guide the optimization of ligands for enhanced affinity and specificity.

In this section, you will analyze the interactions present within the 6DN3 RNA receptor using the Interactions Toggle and the Protein Interaction Analysis panel. You will also analyze the ligand-receptor interactions using the 2D Ligand Interaction Diagram. The Interaction Toggle in the Workspace Configuration Toolbar allows you to inspect various interactions, often represented with colored dashed lines. The Protein Interaction Analysis panel calculates the interactions between two user-defined sets within specified distance threshold for each interaction type. The Ligand Interaction Diagram allows you to visually analyze the ligand-receptor interactions. For more information, please refer to the corresponding documentation.

5.1 Analyze the interactions present within the receptor

Figure 5-1. Visualizing the interactions within the RNA structure.

  1. Right-click the Interactions Toggle in the bottom right corner.

 

By default, only ligand-receptor interactions are shown to avoid cluttering the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. In order to investigate the interactions within the riboswitch, change the settings as follows:

 

  1. In the Interactions Toolbox, change the display from Ligand-receptor to All for Non-covalent bonds and Pi interactions.
    • The interactions are now shown as colored dashed lines.

Note: You can specify arbitrary atom sets (such as custom selection sets) between which to show interactions.

Figure 5-2. Applying labels to each residue.

Adding labels to each residue can help you stay oriented in the structure.

 

  1. Go to the Style Toolbox, click the Apply Labels arrow and choose Residue Information.
    • Take your time to visualize the interactions.

 

Note: You can hover over the residues and see the atoms involved in Hydrogen bonding in the Status Bar to manually inspect the various types of interactions present in the structure.

Each monomeric nucleotide contains aromatic, charged and polar groups resulting in diverse interactions including base-base, base-backbone, and backbone-backbone interactions. Among the non-covalent interactions, edge-to-edge hydrogen bonding i.e. base-pairing (yellow dashed lines) and π-π stacking between nucleobases i.e. base-stacking (blue dashed lines) are present throughout the 6DN3 structure. Both these attractive interactions play a very important role in stabilizing 3D-RNA structures.

 

Examples of base-base interactions include Watson-Crick pairings such as Y:U 94 with X:A 29. Base-backbone interactions consist of base-phosphate interactions like X:C 25 with X:U 24 as well as base-sugar interactions like Y: U 75 with X: U 37. Backbone-backbone interactions involve sugar-phosphate interactions like Y:U 75 with Y: A 73, and sugar-sugar interactions like Y:G 91 with X:U 24.

Figure 5-3. Opening the Protein Interaction Analysis panel from Tasks.

To view the list of all interactions with the respective distances, you can use the Protein Interaction Analysis panel. The interactions are evaluated between two user-specified atom sets.

 

  1. Find and select Protein Interaction Analysis from Tasks.
    • The Protein Interaction Analysis panel opens.

Figure 5-4. Specifying atom sets for analyzing interactions.

By now, you may have noticed that residues within chain X interact both with other residues in chain X and with residues in chain Y. Likewise, residues in chain Y interact both with residues in chain X and with other residues in chain Y. To show all these combinations, both atom sets must include the complete structure.

 

  1. In the Interaction sets section, for Define sets by, select Chain.
  2. For Set 1, click Add and select chains X and Y.
  3. For Set 2, click Add and select chains X and Y.

 

The distance thresholds can be customized depending on the context of analysis. The default cut-off for the maximum H-bond distance is quite strict, so we’ll increase it to cover the mean donor-acceptor distance in nucleic acids.

 

  1. Click Advanced Options.
    • A dialog box opens.

Figure 5-5. Modifying the cut-off distance for Hydrogen bonds.

  1. For Hydrogen Bonds, set the Maximum distance to 3.0 Å.
  2. Click OK.

Figure 5-6. Calculating and visualizing the interactions.

  1. Click Calculate.
    • This calculation takes a few seconds.
    • The Interactions table is populated.
  2. Click on the Distance heading to sort the table by distance.
    • Take your time to scroll and view the interactions in the Specific Interactions column.
  3. Check the Fit on Select option.
  4. Click on a table row to visualize the corresponding interactions in the 3D Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Take your time to examine the interactions in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed thoroughly and identify any non-canonical base pairs within the 6DN3 structure.

 

Note: You can export the Interactions table as a CSV file by clicking on the Export Table option.

 

  1. Close the Protein Interaction Analysis panel.

In addition to the canonical Watson-Crick base pairs, non-canonical base pairs are also present in the 6DN3 structure. For example, see the Wobble Pair Y:U 111 with X:G 2, the reverse A-U Hoogsteen pairs Y:A 92 with Y:U 89, X:A 40 with X:U 37, Y:A 73 with Y:U 69, and the G-A sheared pairs X:G 28 with X:A 14, Y:A 104 with X: G 9, Y:A 90 with X:G 19. These non-canonical base pairs contribute to the formation of kinks, turns, and other structural motifs that support the global fold or bind a ligand or protein.

 

Please note that currently, manual inspection is required to identify canonical and non-canonical interactions as there is no automated method available to identify these interactions directly.

5.2 Analyze receptor-ligand interactions

Figure 5-7. Opening the Ligand Interaction Diagram from Tasks.

Now you will visualize the interactions present between the 6DN3 FMN riboswitch receptor and BRX1555 ligand in the 2D Ligand Interaction Diagram.

 

  1. Find and select Ligand Interaction Diagram from Tasks.
    • The 2D Ligand Interaction Diagram opens.

 

Figure 5-8. The Ligand Interaction Diagram.

  1. Click the Configure View (settings) icon and choose LID Legend.
    • Take your time to visually analyze the ligand-receptor interactions.  

Note: You can click the Clean up button (green tick mark) to generate a less-cluttered orientation of the diagram.

As seen in the Ligand Interaction Diagram, the isoalloxazine ring system of the BRX1555 ligand is stacked (intercalated) between X:A 48 and Y:A 85. The uridine-like moiety forms a pseudo-Watson-Crick base pair with Y:A 99. The phenyl moiety at the end of its alkyl chain is stacked against Y:G 62.

Figure 5-9. The Save Image option to export the Ligand Interaction Diagram as an image.

Optional: Click the Export icon and choose Save Image to export the Ligand Interaction Diagram as an image. For visual clarity, we recommend modifying the resolution while saving the image.

 

  1. Close the Ligand Interaction Diagram.

6. Conclusion and References

In this tutorial, you learned how to visualize nucleic acid structures in Maestro using the Interactions toggle and Protein Interaction Analysis panel. You also learned how to visualize receptor-ligand interactions using the 2D Ligand Interaction Diagram.

You can now for example use SiteMap to deepen your understanding of the receptor pocket, or use the insights into the receptor structure and ligand interactions for building a docking model or optimizing the ligand further. See the further learning section below or the Oligonucleotide Modeling learning path for additional resources.

7. 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