Absolute Binding Free Energy Perturbation to Post-process Docking Results

Tutorial Created with Software Release: 2025-4
Topics: Free Energy Perturbation (FEP), Hit Discovery, Hit-to-Lead & Lead Optimization, Small Molecule Drug Discovery
Products Used: FEP+

Tutorial files

43.4 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 displayedthe 3D display area in the center of the main window, where molecular structures are displayed
Abstract:

In this tutorial, you will learn how to use Absolute Binding Free Energy Perturbation (AB-FEP+) calculations to enrich virtual screening results. In the paper referenced, a virtual screen was performed on the JH2 binding pocket of JAK2 using 3.8 M drug-like compounds from the ZINC15 database (Metadynamics as a Postprocessing Method for Virtual Screening With Application to the Pseudokinase Domain of JAK2). The top 1000 ligands were analyzed with additional methods and 27 compounds with docking scores comparable to 13 known inhibitors were purchased. Assays showed that the compounds had no detected binding to JAK2. Here, you will set up and examine AB-FEP+ results for post-process docking results of non-congeneric ligands to enable enrichment of virtual screening results.

 

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Note on Protein Preparation, Ligand Preparation, Docking, and Custom Force Field Parameters

  1. Setting up Absolute Binding Free Energy Calculations

  1. Analyzing AB-FEP+ Results

  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 automatically 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 table 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/ab-fep_jak2.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. Saving the project.

  1. Go to File > Open Project > AB_FEP_JAK2.prjzip.
    • Structures are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  2. In Save scratch project warning box, click OK.
  3. Go to File > Save Project As.
  4. Change the File name to JAK2_AB_FEP.
  5. Click Save.
    • The project is named JAK2_AB_FEP.prj.

 

Note: 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.

2. Note on Protein Preparation, Ligand Preparation, Docking, and Custom Force Field Parameters

Structure files obtained from the PDB, vendors, and other sources often lack necessary information for performing modeling-related tasks. Typically, these files are missing hydrogens, side chains, and/or whole loop regions. In order to make these structures suitable for modeling tasks, we use the Protein Preparation Workflow to resolve issues. Similarly, ligand files can be sourced from numerous places, such as vendors or databases, often in the form of 1D or 2D structures with unstandardized chemistry. LigPrep can convert ligand files to 3D structures, with the chemistry properly standardized and extrapolated, ready for use in virtual screening.

In this tutorial, the protein and ligands have already been prepared in order to save time. However, these preparation steps are a necessary part of a virtual screen and must be done before any FEP+ calculations. Please see the Introduction to Structure Preparation and Visualization tutorial for instructions on using the Protein Preparation Workflow and Preparing Protein and Ligand Structures for FEP+ for tips on structure preparation for FEP+. These structures are included in the project in the Receptors group. Additionally, the prepared ligands are available in the group ligprep_JAK2-out1.

The ligand poses for this calculation were determined using Glide SP docking into the ATP binding site of the JAK2 structure 4FVR. The ligand was removed and a receptor grid was generated using the methods listed in Metadynamics as a Postprocessing Method for Virtual Screening With Application to the Pseudokinase Domain of JAK2. The top-scoring pose for each ligand is in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion as the group JAK2_10ligands_pv. While the binding poses for the WC1 and WC2 agree with the crystal structures (5USZ and 5UT0, respectively), the docking scores do not correlate to the Kd values of the three known binders, JAK179, WC1, and WC2. This is expected, as docking scores are not designed to correlate with binding affinity measurements. However, to help enrich virtual screening results, it is helpful to have a better sense of how ligands with unknown binding affinity compare in a virtual screen to known binders. We will use AB-FEP+ to post-process these docking results to more accurately reflect the experimental results.

Additionally, this ligand set uses custom force field parameters which are provided for you in the directory ffb_JAK2_oplsdir. You can point to these custom parameters by going to your Maestro Preferences and pointing to your Working Directory to choose the ffb_JAK2_oplsdir. More information for using custom force field parameters can be found here.

3. Setting up Absolute Binding Free Energy Calculations

In this section, you will import docking results into the FEP+ panel to set up the calculation. The docking output file used contains both prepared ligands and a prepared protein. Note that the ATP has been removed from the 4FVR structure for docking and for FEP+ calculations.

Figure 3-1. Selecting the docking output.

  1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, 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 the JAK2_10ligands_pv group by clicking on the group heading.
    • All group entries are 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.
  2. In the Tasks, find and select FEP+.
    • The FEP+ panel opens.

Figure 3-2. Importing structures from the Project Table and choosing to calculate the Absolute binding free energy.

  1. For Import structures or perturbation map from, choose Project Table (11 selected entries).
  2. Click Import.
    • The receptor is automatically detected.
    • Options for how to calculate the Binding Free Energy are shown.
    • A new group FEP+: 4FVR_prepared_FEP is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion with the top entry 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.

Figure 3-3. Hovering over the warning symbol to get more information.

 

 

 

Figure 3-4. Clicking on the warning symbol to view the Protein Reliability Report for the 4FVR receptor.

Here, the issue with the receptor is for steric clashes stemming from the crystal mates. As all these clashes are a sufficient distance from the binding site, we will not worry about them.

 

Note: If there is a warning for your ligands, we strongly recommend running the Force Field Builder to calculate any missing torsions.

Figure 3-5. Choosing to calculate the Absolute binding free energy.

  1. Next to Calculate Binding Free Energy for, choose Absolute.
    • The information is populated into the FEP+ Overview tab.
    • A 2D version of the ligands are shown in the table and checked for quality.

 

 

 

Figure 3-6. The Overview tab.

Note: The green checks in the Quality column indicate the torsions for the ligand are described by the force field. If a warning symbol is in the Quality column, hover over it to get more information. See Troubleshooting Common Issues for more details.

Figure 3-7. Ignoring the warning in the info section.

Note: You can ignore the warning in the Info section about the missing .fmpdb file, since we are only setting up the calculation.

Figure 3-8. Choosing to add the experimental affinity data.

  1. In the Overview tab, click Affinity and choose Experimental Data.
    • The Choose Affinity Property dialog box opens.

Figure 3-9. Adding in the affinity data.

  1. In the Choose Affinity Property panel, choose the Kd [uM] (User).
  2. Next to Affinity units, choose Ki (µM).
  3. Click OK.
    • The affinity data is added to the FEP+ panel.

Figure 3-10. Renaming the job and modifying the job settings.

  1. Change the Job name to absolute_binding_JAK2.
  2. Click the Job Settings (cog).

Figure 3-11. Setting the CPU and GPU hosts.

  1. Choose your CPU Host and GPU Host.

 

Note: Ensure Maximum simultaneous subjobs are set to 0. This removes the limit on the number of subjobs, so they are all submitted to the subjob host queue. If you do not have license checking enabled, set the number of subjobs to ensure that you do not exhaust your licenses. Optionally, set the total number of GPUs to be used.

 

Note: This job requires significant GPU resources to run, so you will look at pre-generated results.

 

  1. Optional: Click Run to start the job.

 

Note: Depending on the nature of the ligands being assessed, receptor size, and the type of GPU being used, you may need to use more than 1 GPU to prevent the calculation from running out of memory.

Figure 3-12. Writing the job input files and a shell script to launch the job.

Note: For a job of this length, we would recommend writing out the input files and launching the job from the command line, versus launching from the GUI. For more information on how to do this, please see the FEP+ Command Reference Manual.

4. Analyzing the AB-FEP+ Results

Figure 4-1. Importing the AB-FEP+ results into the project.

  1. Find and open FEP+ from Tasks.
    • The FEP+ panel opens.
  2. For Import structures or perturbation map from, choose File.
  3. Click Browse and choose absolute_binding_JAK2_out.fmp.
  4. Click Open.
    • The file is loaded into the panel.
    • The structures are added to a new group in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  5. In the FEP+ panel, click Next.

Figure 4-2. Reordering by the Predicted Error.

  1. In the FEP+ panel, click the Pred. Binding ΔG column header.
    • The table is reordered with the lowest values at the top.
    • The Bennett error values are shown in kcal/mol next to the predicted ΔG values.  

Note: Predicted Error values of < 0.3 kcal/mol are good, values > 0.3 kcal/mol indicate that some troubleshooting may be needed. For instance, repeating simulation using a different random seed or running the simulation for longer may address the underlying issue.

Figure 4-3. Viewing the Analysis tab.

  1. Go to the Analysis tab.  

Note: If the error for an edge is > 0.3, please see Troubleshooting Common Issues for more information.

  1. In the row for JAK199, click View.
    • The Analysis panel opens in the Protein-Ligand Interactions tab.

Figure 4-4. Observing the high RMSD fluctuation for the JAK199 ligand.

  1. Go to the Ligand Details tab.
  2. Click Properties.
    • There is large fluctuation in the RMSD of the ligand at the end of the simulation.
    • This indicates the binding mode may not be stable.
    • As the predicted ΔG is low compared to known binders (-7.6 compared to JAK179’s -16.0), this unstable binding mode is not unexpected.
  3. Close the Analysis panel.

Figure 4-5. Analyzing the JAK179 ligand.

  1. In the FEP+ panel, for the row for JAK179, click View.
    • The Analysis panel opens.

Figure 4-6. Observing the unstable convergence for the Complex Leg.

  1. Go to the Convergence tab.
    • The convergence of the complex leg may benefit from a longer simulation.  

Note: The y-axis of the convergence plots will adjust to fit the scale. Pay attention to the y-axis values as good convergence may appear poor if the scale is very small.

  1. Close the Analysis panel.

Figure 4-7. Reviewing the complex trajectory for the JAK179 ligand.

  1. In the FEP+ panel, for the JAK179 ligand under Complex Trajectory, click 5.0 ns.
    • A new group is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
    • The new entry 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.

Figure 4-8. Loading the trajectory.

  1. Click the T button and choose Load Trajectory.
    • The Trajectory Viewer loads into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-9. Hiding the solvent atoms.

  1. In Quick Select, click S.
    • The solvent atoms are 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 Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Open the Style toolbox and click Undisplay selected atoms (closed-eye icon).
    • The solvent atoms are now hidden in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-10. Adjusting the trajectory Playback Settings to reduce the speed and hide atoms beyond the binding site.

  1. In the Trajectory Viewer, click Playback Settings.
  2. In the Basic tab, next to Speed, reduce to 80%.
  3. Check Beyond binding site to hide these atoms.

Figure 4-11. Adjusting the trajectory Playback Settings to align playback on the protein.

  1. Click the View Position tab.
  2. Next to Align on, choose Protein.
  3. Click in the empty Workspace to close Playback Settings.
  4. Type L to zoom to the ligand.

Figure 4-12. Playing the complex trajectory.

  1. In the Trajectory Viewer, click Play.
    • The frames from the 5 ns complex simulation are shown.
    • There is a lot of rotation of the carboxylic acid and phenyl group on either side of the sulfone.

Figure  4-13. Observing the different orientations of the sulfonamide and the carboxylic acid groups.

  1. Click Move one step forward to manually view the frames.
    • The orientation of the sulfonamide and carboxylic acid change several times.
    • These groups, particularly the sulfonamide, are solvent-exposed so higher mobility is expected.
    • The binding pose is stable across the simulation.

Figure 4-14. Opening the Project Table.

  1. Open the Project Table.

Figure 4-15. Hiding all the properties in the Property Tree.

  1. In the Property Tree, double-click the All check box.
    • All property rows are hidden.

Figure 4-16. Displaying the dg properties.

  1. Search for dG.
  2. Check the box for exp dG and pred dg values.

Figure 4-17. Sorting on the pred dg property.

  1. Right-click on the pred dg values column header.
  2. Choose Sort All (Ascending).
    • AB FEP correctly identifies WC1 and JAK179 as tight binders, WC2 as a weaker binder, and the rest of the ligands as non-binders.
    • While the exact value of binding affinity differs, the rank ordering agrees with experimental measurements.

5. Conclusion and References

In this tutorial, you used Absolute Binding Free Energy calculations to evaluate a series of non-congeneric ligands. Results from the Absolute Binding Free Energy calculation were used to re-rank the ligands.

6. 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

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 that files are saved

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