Protein FEP for Ligand Selectivity Panel

Evaluate the change in binding free energy on mutation of protein residues in the presence of a ligand to determine whether the residues are important for ligand selectivity.

To open this panel: click the Tasks button and browse to Free Energy Perturbation → Protein FEP for Ligand Selectivity.

  • Using
  • Features
  • Additional Resources

Using the Protein FEP for Ligand Selectivity Panel

The purpose of this panel is to provide information on how the mutation of protein residues affects the binding of a ligand. It can be used to probe drug resistance and explore the role of various protein residues in ligand binding. As such, it is a complement to the FEP+ tool, which can be used to explore the variation of ligand structure on binding.

The input is a protein structure (with or without a membrane) and one or more ligand structures, though only a single ligand can be used currently. As for FEP+, the structures are immersed in water for the simulations, with enhanced water sampling via GCMC. You can add a POPC membrane if needed by selecting Run membrane protocol in the FEP+ Advanced Options Dialog Box. The output structures for each mutation are written to a Maestro file.

Submitting a structure with incorrect PDB names results in those names being overwritten, as proper PDB names are required for atom mapping.

To choose a residue to mutate, you can pick it in the Workspace, pick it in the ligand interaction diagram, or select it in the residues table. When you have chosen a residue, click in the Mutation column to set the mutations. You can mutate multiple residues, each with multiple mutations.

FEP calculations can use a large amount of memory. There is a script that you can use to estimate the memory required for an FEP job—see fep_memory_estimation.py Command Help for more information. You can estimate the number of atoms by building the initial system, including solvent, in the System Builder Panel. With a solvent buffer of 8 Å, the output structure approximately provides an upper limit to the size of the FEP systems. This information is needed to estimate the memory required.

When the job is run, you can examine the results by opening the results .fmp file in the FEP+ Panel ( click the Tasks button and browse to Free Energy Perturbation → FEP+). In the FEP+ Analysis Tab you can view all the results and display the trajectories.

Protein FEP for Ligand Selectivity Panel Features

Step 1: Import structures from buttons

Import the protein and ligand structures from the source defined by the buttons. The name of the receptor and the number of ligands are reported below the buttons on import.

  • Project Table—Use the entries that are currently selected in the Project Table. The ligands must be selected in the Project Table. The protein must either be the first selected entry in the Project Table, or be included in the Workspace (and not selected). The ligands must be in separate entries: if the protein has a ligand in the active site, it is ignored.
  • File—Use the structures from the specified file. Clicking the button opens a file selector, so you can navigate to the file you want to open. The file must be a Maestro file, and contain the protein in the first entry and the ligands in the rest ("pose viewer" format). The structures in the file are imported into the Maestro project.
Step 2: Select a ligand for residue mutation calculation table

Lists available ligand structures and indicates whether calculations can be run with these ligands, in the Health column. You can select a single ligand from the table, which will be used for the calculations. When you select a ligand, it is shown in the ligand interaction diagram. The tool tip in the Health column gives information about problems with the ligand that would prevent a calculation from being run. The main problems to be addressed are listed below:

Problem Solution
Steric clashes Check the input conformation
Torsion parameter missing Generate new parameters with Force Field Builder
Lewis structure faulty Run bond order assignment on the ligand
Hydrogens missing Add hydrogens to the ligand

The last two issues can be fixed by running LigPrep on the ligand (which is recommended).

Step 3: Select residues to mutate controls

These controls allow you to show a filtered list of the residues in the protein, select the residues you want to mutate, and choose the mutations for each residue. The total number of mutations is displayed and updated as you choose mutations. The selections you can make depend on whether you want to perform single mutations on a protein to produce a set of mutants (in the Single mutations tab), or whether you want to perform multiple simultaneous mutations on a protein to generate one or more mutants (in a Multi-site mutation tab for each protein).

Residue scan with λD option

Select this option to run FEP+ residue scanning with the lambda dynamics approach, which can rapidly identify high quality protein variants [15]. There are certain limitations to using this option:

  • The mutation chain must be a valid, isolated substructure.
  • Multi-site mutations are not supported.
  • Mutation to/from the amino acid Proline is not supported.
  • Non-Standard Amino Acids (NSAA) are not supported.

Advanced options for this option can be selected from the λD Advanced Options Dialog Box.

For a tutorial, see Identifying impactful mutations using FEP+ residue scanning.

Single mutations tab

Select sites and mutations to generate a set of mutant proteins with a single mutation from the parent protein. For each site, a mutated protein is generated for each selected mutation.

Show option menu

Show residues in the table below that match the criterion chosen from this option menu. The choices are all residues, or residues within 4Å of the ligand.

Pick residue option

Pick a residue in the Workspace to mutate. The residue is selected in the table.

Residues table

This table lists the residues in the protein, filtered by the choice from the Show option menu. Both standard and nonstandard residues can be mutated. You can select a row in the table to mutate or pick a residue in the Workspace. If the protein structure contains other molecules that have residue names, these are listed in the Mutation column as Prohibited. You can mutate multiple residues: setting the mutations for these residues must be done one residue at a time, however. If you want to mutate to nonstandard residues, you must select them first in the Nonstandard Residues Panel. A count of residues selected for mutation and total mutations is shown below the table.

Click in the Mutation column to choose the mutations for a residue. A small pane opens above or below the row. The small pane closes when you click Close, click elsewhere in the main panel, press Esc or Enter, or move the pointer out of the main panel.

In this pane you can select multiple mutations, either by individual residue or by residue type (click Select to choose the residue type). You can select all of the standard or nonstandard residues (or both) by checking the relevant box. A gray square indicates partial selection of a category. Each selection adds to the list shown in the table cell. You can use the search field to filter the list of nonstandard residues. The structure of a nonstandard residue is shown in a tooltip when you pause the pointer over the residue name. The check boxes for some residues may be disabled if they may not be selected as valid mutations for the current residue: this is the case for mutations between proline and charged residues. Click Clear All to clear the selection.

Multi-site mutation tab

Select sites to mutate and a single residue for each site to mutate to, to generate a protein with multiple mutations. Only one mutation can be made at a given site. To define another multi-site mutant of the protein, click the + tab to the right, to add another copy of the Multi-site mutation tab in which to define the mutations.

Show option menu

Show residues in the table below that match the criterion chosen from this option menu. The choices are all residues, or residues within 4Å of the ligand.

Pick residue option

Pick a residue in the Workspace to mutate. The residue is selected in the table.

Residues table

This table lists the residues in the protein, filtered by the choice from the Show option menu. Both standard and nonstandard residues can be mutated. You can select a row in the table to mutate or pick a residue in the Workspace. If the protein structure contains other molecules that have residue names, these are listed in the Mutation column as Prohibited. You can mutate multiple residues: setting the mutations for these residues must be done one residue at a time, however. If you want to mutate to nonstandard residues, you must select them first in the Nonstandard Residues Panel. A count of residues selected for mutation and total mutations is shown below the table.

Click in the Mutation column to choose the mutation for a residue. A small pane opens above or below the row. The small pane closes when you click Close, click elsewhere in the main panel, press Esc or Enter, or move the pointer out of the main panel.

In this pane you can select a single residue for the mutation. You can use the search field to filter the list of nonstandard residues. The structure of a nonstandard residue is shown in a tooltip when you pause the pointer over the residue name. The check boxes for some residues may be disabled if they may not be selected as valid mutations for the current residue: this is the case for mutations between proline and charged residues. Click Clear to clear the selection.

Ligand interaction diagram

This diagram shows a 2D image of the selected ligand and the nearby protein residues, with interactions marked. You can click on a residue in the diagram to select it in the residues table, which also selects it as a residue to mutate. For more information, see the Ligand Interaction Diagram Panel topic.

Job toolbar

Manage job submission and settings. See Job Toolbar for a description of this toolbar.

The FEP+ tasks are run on the GPU host chosen in the the Job Settings Dialog Box. Typically, three subjobs are submitted per perturbation (for complex, solvent, vacuum). You can set Maximum simultaneous FEP subjobs to limit the number of subjobs on the GPU host at the same time.

The Settings button menu provides access to custom features:

  • Advanced Options—set advanced options for the MD simulations. Advanced options cannot be set when uploading to FEP+ Web Services. Opens the FEP+ Advanced Options Dialog Box.

If you have purchased Web Services, the Submit to Web Services option is present. Select to upload the job to Web Services for execution, and click the Submit button. The number of credits to be used and the number of available credits are reported, and the job is uploaded on confirmation of the credit usage for the job. If you have access to multiple Web Services projects, you are prompted to choose the project to associate the job with. The FEP+ Web Services Job Table Panel opens.

If you have Web Services credentials but do not see the Submit to Web Services option, see Troubleshooting.

The Job Settings button opens the Protein FEP for Ligand Selectivity - Job Settings Dialog Box, where you can make settings for running the job.

Status bar

The status bar displays information about the current job settings and status for the panel. The settings includes the job name, task name and task settings (if any), number of subjobs (if any) and the host name and job incorporation setting. The job status can include messages about job start, job completion and incorporation.

Use the Reset button to reset the panel to its default settings and clear any data from the panel. You can also reset the panel from the Job toolbar.

The status bar also contains the Help button , which opens the help topic for the panel in your browser. If the panel is used by one or more tutorials, hovering over the Help button displays a button, which you can click to display a list of tutorials (or you can right-click the Help button instead). Choosing a tutorial opens the tutorial topic.

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