MM-GBSA Residue Scanning Panel

In this panel you can scan a protein for residues that are candidates for mutation, using homology modeling to identify variable or conserved residues, solvent-accessible surface area to identify surface residues, distances to identify contacts with other residues, surface shape complementarity, and manual selection. The mutations can then be defined and applied to produce mutated structures. By default, mutations are applied independently to produce a set of structures with single mutations. This process can be repeated automatically to optimize the stability of the protein or the affinity of two parts of the protein for each other. Mutations can also be applied simultaneously.

To open this panel, click the Tasks button and browse to Biologics → MM-GBSA Residue Scanning Calculations.
To open this panel from the entry group for the results of an antibody structure prediction job, use the Workflow Action Menu .

  • Using
  • Features
  • Additional Resources

Using the MM-GBSA Residue Scanning Panel

Some examples of the use of residue scanning are:

  • improvement of binding affinity or protein stability
  • identifying protein-protein interface hotspots
  • identifying residue mutations that can improve stability
  • mutating unpaired, solvent-exposed Cys residues to reduce undesired reactivity

The basic workflow is as follows:

  1. Prepare the protein for modeling using the Protein Preparation Workflow Panel, if it is not already prepared.

  2. Import the structure into the workflow, either from the Workspace or from a Maestro file (Import structure from).

  3. Choose the calculation type. Stability is always calculated, affinity can be calculated if there is more than one chain.

  4. If you chose to calculate affinity, select the chains for one of the partners in the binding affinity calculation.

  5. Choose the mutations for the residues you want to mutate. You can do this manually or by homology criteria—see Defining residue mutations for instructions.

  6. If you want to optimize the stability or binding affinity, select Perform affinity maturation in the Options tab, and choose an optimization procedure.

  7. Choose Job Settings from the Settings button menu,

    set the job parameters in the Job Settings dialog box, and click Run to run the job; or click Run to run the job with the current job settings.

A structure is produced for each mutation, and the structures are incorporated into the project as new entries when the job finishes. As the time taken scales linearly with the number of mutated structures produced, you should consider carefully how many mutations you make. The number of mutation sites and the total number of mutations (structures generated) is reported below the table.

As part of the calculation, the change in a number of properties is calculated, some of which are optional. These properties are displayed when you view the results. The change in property value that is displayed is the value for the mutated structure minus the value for the parent structure, so a positive value for the change means that the mutated structure has a larger (more positive) value than the parent structure. The change in stability is always calculated, and also the solvent-accessible surface area (SASA), hydropathy, rotatable bonds, and complementarity. The properties are described in more detail in Table 1

You can also export mutations from the Protein Interaction Analysis Panel to this panel; if you do this, you must import the same structure as used for the protein interaction analysis, and choose to calculate affinity.

Residue scanning for antibodies can be done in the Antibody Humanization: Residue Scanning panel, which uses antibody databases to search for homologs in the antibody itself. The Residue Scanning panel offers more general residue mutation capabilities, which can also be used for antibodies—for example, if you want to mutate both the antibody and the antigen simultaneously.

The method used for residue scanning tends to bias toward arginine and methionine substitutions. It is thus recommended to verify that the predicted favorability of methionine or arginine mutations can be rationalized by visual inspection of the associated 3D structures.

To run residue scanning from the command line, you can use the following command. Run the command with -h for more information.

$SCHRODINGER/run residue_scanning_backend.py

To write out input files, click the arrow next to the Settings button,

and choose Write (more...).

For information on command options, see residue_scanning_backend.py Command Help.

If any of the subjobs fail, you can rerun them by using the --merge_mae command option.

To visualize the results of the residue scanning calculation, you can use the MM-GBSA Residue Scanning/Humanize Antibody Viewer Panel (Biologics → MM-GBSA Residue Scanning Results). To open this panel from the entry group for the results of a job, and load the results, use the Workflow Action Menu . The panel opens automatically when the job is incorporated into the Maestro project.

Defining residue mutations

Instructions for defining residue mutations for various cases are given below. For manual selection, a residue selection pane is displayed. This tool lists the 22 standard residues, and provides a selection tool to select groups such as Aromatic, Aliphatic, Polar, Nonpolar. If you have nonstandard residues defined, these are listed under the standard residues and a Nonstandard type is added to select them all. You can select more than one residue or group. For information on defining nonstandard residues, see Nonstandard Residues Panel.

To define the mutations for a single residue:

  1. Click in the Mutations column for the residue.

    A popup pane is displayed below the table cell.

  2. Choose the residues from the popup.

  3. Press Enter to finish adding mutations and dismiss the popup.

    The mutations are now listed in the table cell.

    If you want to cancel the changes and dismiss the popup, press ESC.

To select a set of residues to define mutations for, you can do one of the following:

  • Select the table rows for the residues.

  • Select Pick Workspace residue and pick residues in the Workspace.

  • Choose Residue selection from the Mutate option menu, select options for limiting the types of residues to be selected in the Residue Scanning - Select Residues panel, and click OK.

  • Choose Atom Selection and use the selection tools in the Atom Selection dialog box to select the residues, and click OK.

To apply a set of mutations to selected residues:

  1. Select the residues in the table that you want to apply the same mutations to.

  2. Choose Selected residues from the Mutate option menu.

  3. Click the to tool (next to the Mutate option menu).

    The residue selection popup is displayed below the tool.

  4. Choose the residues from the popup.

  5. Press Enter to finish adding mutations and dismiss the popup.

    The mutations are now listed on the text box of the to tool.

    If you want to cancel the changes and dismiss the popup, press ESC.

  6. Click Apply.

    The mutation list is set for the residues that you selected. The mutations are listed in the Mutations column of the table.

To apply a set of mutations to all residues:

  1. Choose All residues from the Set mutations for option menu.

  2. Click the to tool (next to the Set mutations for option menu).

    A popup panel is displayed below the tool. The 22 standard residues are listed, along with groups such as Aromatic, Aliphatic, Polar, Nonpolar. If you have nonstandard residues defined, these are listed under the standard residues and a Nonstandard type is added to select them all. You can select more than one residue or group.

  3. Choose the residue types from the popup.

  4. Press Enter to finish adding mutations and dismiss the popup.

    The mutations are now listed on the text box of the to tool.

    If you want to cancel the changes and dismiss the popup, press ESC.

  5. Click Apply.

    The mutation list is set for all residues in the Mutations column of the table.

To apply a set of mutations using homology criteria:

  1. Click Homology Criteria.

    The Homology Suggestions dialog box opens.

  2. Run a search for homologs (Run a Blast Search) and align the homologs (Align Homologs).

  3. Specify the criteria for automatic selection of residues, in the Selection by homology criteria and Parent structure 3D criteria sections.

  4. Click Select in the Residues Tab.

    The Residues tab is displayed in the main panel and the residues are selected in the table. The default selection of mutations is based on the variations seen in the homologs.

MM-GBSA Residue Scanning Panel Features

Import tools

The import tools allow you to import a protein structure into the workflow for residue mutation. You can choose an option from the Import structure from option menu to use the entire structure in the Workspace, the selected residues in the Workspace, or a structure from a Maestro file. For the first two options, click the Import button to import and analyze the structure. If importing from a file, the button changes to Browse, and you can click this button to open a file selector to locate and open the file. If a residue has the same structure (detected by SMILES) as an existing residue in the same set, the import of that duplicate residue will be skipped, and the import summary dialog will list this residue and the reason why it was skipped.

Calculation type options

These options allow you to choose between calculating the protein stability as a result of mutation (Stability), or to calculate both the stability of the protein and the affinity of one part of the protein for another (Stability and Affinity).

Stability option

Calculate the stability of the protein, in energy units. This is the default, and is the only option available if the protein has only one chain.

Stability and Affinity option

Calculate the stability of the protein and the affinity of one set of chains to the remaining chains, in energy units. If you select this option, the Binding partners tools are displayed so you can define the two parts of the protein in terms of its chains.

Binding partners option menu

Choose the chains to use for one of the two binding partners for affinity calculations from this option menu. The remaining chains are assigned to the other partner, and are listed next to next to binding to chains:.

Import mutations menu

Click on the Import Mutations link to open the menu. In this section, you can choose to import mutations using Homology criteria or via a Mutations file by clicking their respective links:

Homology criteria option

Select residues to mutate using suggestions from homologous proteins, which are based on variability or conservation at residue positions, or on solvent accessibility or side-chain interactions. When you click this option and then the corresponding Select button, the Homology Suggestions Dialog Box opens. In the Homology Suggestions Dialog Box you can build a homology model for a chain and select residues by conservation or structural criteria. Clicking Save in this dialog box selects the suggested residues in the residue table of the Residues tab and sets the mutations for the residue to the residues in the homologs that differ from the query.

Mutations file option (beta)

Select to upload mutations from a FASTA file containing mutational variants to pre-populate the residue mutation panel. Click the Browse button to search for the desired sequence file.

Residues tab

In this tab you select the residues for mutation.

Set mutations for option menu

Select the residues whose mutations you want to set up, from this option menu. The mutations are chosen from the to option menu, and are applied to all selected residues. The choices are:

  • All residues—Set the mutations for all residues in the table. When you select this option, all residues are selected in the residues table.

  • Selected residues—Set the mutations for the residues that are selected in the residues table.

  • Residue selection—Set the mutations for selected residue types. Opens the Residue Scanning - Select Residues dialog box, in which you can limit the selection of residues by chain, solvent exposure, or residue type (including nonstandard residues). All residues that meet the criteria are selected in the residues table when you click OK in the dialog box. The setting on the Mutate option menu is switched to Selected residues, as the dialog box is simply a means of selecting residues.

  • Atom selection—Select residues for setting up mutations by general atom selection procedures. Opens the Atom Selection Dialog Box, in which you can make selections by combinations of criteria. All residues that meet the criteria are selected in the residues table when you click OK in the dialog box. The setting on the Mutate option menu is switched to Selected residues, as use of the dialog box is simply a means of selecting residues.

to option menu and Apply button

Select a set of mutations from the popup that appears when you click on the menu, and click Apply to set these mutations for the residues that are selected in the table. The popup allows selection of individual residues, selection by type, and the selection can include nonstandard residues defined by the current nonstandard residues database.

Allow simultaneous mutations option

Mutate residues selected for mutation simultaneously. Each possible combination of mutations is generated. You can select combinations of mutations, or classes of mutations (single, double, etc) by clicking Preview and making selections in the Potential Mutants Dialog Box. When this option is not selected, mutations are performed for each residue separately.

This option is deselected if you select Perform affinity maturation in the Options tab or Apply mutations symmetrically to chains. There is no fixed limit on the number of mutated structures, but a warning about performance is posted if the number of mutated structures is greater than 1000.

Apply mutations symmetrically to chains option and menu

For structures that have multiple identical chains (i.e. is or contains a homomultimer), apply the mutations simultaneously to identical chains. The pairs of chains that you consider to be identical must be chosen from the popup that appears when you click on the menu. This menu lists the pairs of chains with their percentage similarity. As chains that are formally identical can have small variations, this allows you to override these small variations (rather than requiring 100% similarity). It also allows you to mutate only some of the identical chains.

This option is deselected if you select Allow simultaneous mutations, or Perform affinity maturation in the Options tab.

Import mutations menu

Click on the Import Mutations link to open the menu. In this section, you can choose to import mutations using Homology criteria or via a Mutations file by clicking their respective links:

Homology criteria option

Select residues to mutate using suggestions from homologous proteins, which are based on variability or conservation at residue positions, or on solvent accessibility or side-chain interactions. When you click this option and then the corresponding Select button, the Homology Suggestions Dialog Box opens. In the Homology Suggestions Dialog Box you can build a homology model for a chain and select residues by conservation or structural criteria. Clicking Save in this dialog box selects the suggested residues in the residue table of the Residues tab and sets the mutations for the residue to the residues in the homologs that differ from the query.

Mutations file option (beta)

Select to upload mutations from a FASTA file containing mutational variants to pre-populate the residue mutation panel. Click the Browse button to search for the desired sequence file.

Mutations table options menu

Click the gear icon to open the menu and display the options for the mutations table. The Sync with Workspace option, enabled by default, allows you to select residues from the Workspace, with selections reflected in the table also. You may also toggle Fit on select, as well as the display of chosen columns in the Show columns menu.

Residues table

Table containing a list of all the residues in the Workspace that were included in the analysis, and so can be considered for mutation. This table can be used to define and list the mutations for each residue. You can select multiple rows in the table, for example to set a common set of mutations. The number of residues selected and the total are reported below the table. Residues that are selected in the table are also selected in the Workspace. The table lists properties of the residues that may be useful for choosing residues to mutate.

Chain Chain name. When mutations are applied symmetrically to multiple chains, the chains are listed here, e.g. A,B,C. If one of these chains does not have a particular residue, it is not included in the list, e.g. if only chain B has residues 1 and 2 and chains A and C start at residue 3, this column would contain only B for residues 1 and 2.
Residue Residue number and insertion code, and 3-letter residue name.
Mutations List of mutations that will be applied to the residue. If you click in the column, a small pane is displayed, from which you can choose the mutations.
In this pane you can select multiple mutations, either by individual residue or by residue type (click Select to choose the residue type). To mutate to nonstandard residues, select them first in the Nonstandard Residues Panel; they are listed below the standard residues. You can use the search field to filter the list of nonstandard residues.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. You can select all of the standard or nonstandard residues (or both) by checking the relevant box. Click Clear All to clear the selection. 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.
Disulfide Indication of whether the residue forms a disulfide bond with another residue.
#Pi-pi Number of pi-pi interactions formed by the residue.
#H-bonds Number of hydrogen bonds formed by the residue with other molecules (other chains, ligands, waters, cofactors).
Buried Percentage of the solvent-accessible surface area of the residue that is buried due to the presence of other chains. If the residue is not at the interface or there is only one chain, the column contains the text "NA", for "not applicable".
Surface Complementarity If the residue is at the interface between two chains, the surface complementarity of the residue to residues in the other chain is evaluated and displayed in this column. The surface complementarity measures how well the shape of one surface matches the shape of the other. It is calculated from the scalar product of the normal to one surface at each surface point and the normal to the other surface at the point nearest to the first surface, with a weighting factor for the distance between the points. The score is the normalized sum over surface points, such that perfect complementarity (parallel normals) has a value of 1, and no complementarity has a value of 0. It is evaluated over the buried solvent-accessible surface area. (See Lawrence, M. C.; Colman, P. M. J. Mol. Biol., 1993, 234, 946–950.)
If the residue is not at the interface or there is only one chain, the column contains the text "NA", for "not applicable".
Fit on select option

Fit the selected residues in the Workspace to the screen size, when residues are selected in the table. The selected residues are colored with green carbons, and all other residues are colored with a darker color. The color scheme remains in effect when you deselect this option.

View mutants button

Preview the list of mutations, and optionally select the mutations to be performed. Opens the Potential Mutants Dialog Box, which lists the mutants in a table. You can select the mutations to perform in several ways. Clicking the View sequences link will open the Multiple Sequence Viewer (MSV) GUI with sequences from the structures and mutated chains selected.

Options tab

In this tab you can set options for refinement of the mutated structure and for optimization of properties of the protein by successive mutation.

Refinement option menu

Choose the method for refinement of the mutated residues and optionally of nearby residues as well.

When residues are mutated, the structure around the mutation site should be allowed to relax in response to the mutation. Before the minimization of the region around the mutation site, a side-chain prediction of the mutated residues is performed, which does a thorough exploration of possible side-chain conformations.

  • Side-chain prediction—Run a Prime side-chain prediction, adjusting only the side-chain rotamers (repacking), then minimizing the side-chain atoms.
  • Side-chain prediction with backbone minimization—Run a Prime side-chain prediction first, adjusting only the side chains, followed by a minimization of the entire residue. This allows relaxation of the entire residue.
  • Side-chain prediction with Cbeta sampling—Run a Prime side-chain prediction that includes variation of the CA-CB direction as well as side-chain rotamer adjustment, followed by minimization of the entire residue.
  • Side-chain prediction with backbone sampling—Run a Prime side-chain prediction that includes backbone adjustments of the atoms between the CA atoms on the adjacent residues, as well as CA-CB adjustments and side-chain rotamer adjustments, followed by minimization.
Cutoff text box

Specify the cutoff distance for including residues in the refinement that are near to the mutated residues. Any residue that has an atom within the specified distance of a hypothetical Arg residue at the mutation site is included in the refinement. A hypothetical Arg residue is used to ensure that the set of residues refined is identical regardless of the initial or mutated residue identities.

Perform affinity maturation option

Perform a series of mutations to optimize either the stability or the binding affinity of the protein. One residue is mutated at a time, and the mutated structure can be mutated again, to produce structures with multiple mutations.

If this option is selected, the Allow simultaneous mutations option and the Apply mutations symmetrically to chains option in the Residues tab are deselected, as these options are incompatible with affinity maturation.

Optimization option menu

Choose the method for optimizing the property. The two choices are:

Monte Carlo optimization

Perform a Monte Carlo optimization of the chosen property, in which mutations are chosen randomly from the set defined for the job. At each Monte Carlo step, one of these residues is mutated at random. Residues that have already been mutated can be mutated again, and mutated back to the original residue. The acceptance criterion for the step (or move) is based on the change in affinity or stability. The process is repeated until the maximum number of steps given in the Maximum steps text box is reached. You can control some aspects of the Monte Carlo procedure with settings in the MM-GBSA Residue Scanning - Advanced Options Dialog Box (click the Advanced button).

Exhaustive enumeration

Perform all possible mutations of the residues to locate the structures with the best affinity or stability. You should keep the number of residues and mutations per residue small if you choose this option, to avoid combinatorial explosion. For example, mutating 5 residues with only one mutation per residue results in 31 possible mutated structures; with two mutations per residue the number of possible mutated structures is 242; with three mutations it is 1023; with four it is 3224; and with ten it is 161050.

Maximum steps text box

Set the maximum number of Monte Carlo steps in this text box. Each step consists of a single mutation, chosen randomly from the set of mutations defined for the job. The acceptance criterion for the step (or move) is based on the change in affinity or stability.

Maximum number of simultaneous deviations from input text box

Set an upper limit on the number of mutated residues in any output structure with respect to the input structure.

Property to optimize options

Choose the property that is optimized in the mutation process:

  • Affinity—Optimize the binding affinity of one set of chains to another set. This option is only available if you select Stability and Affinity for the calculation type, and define two sets of chains for the binding partners.
  • Stability—Optimize the stability of the protein. This option is selected and the options are unavailable if there is only one chain or if the calculation type is Stability.

Both properties are calculated with Prime MM-GBSA in implicit solvent, and are given in kcal/mol. The change in property value that is displayed is the value for the mutated structure minus the value for the parent structure, so a positive value for the change means that the mutated structure has a larger (more positive) value than the parent structure. See Binding Affinity Prediction Method and Stability Prediction Method for more information.

The change in stability is always calculated, and also the solvent-accessible surface area (SASA), hydropathy, rotatable bonds, and complementarity. The properties are described in more detail in Table 1

Maximum number of output structures text box

Specify the maximum number of output structures to return. The structures that are returned are those with the best (most negative) value of the binding affinity or the stability.

Define Nonstandard Residues button

Open a database of nonstandard residues or add new nonstandard residues to a database. Opens the Nonstandard Residues Panel. The nonstandard residue database that is selected in this panel is used for identifying nonstandard residues in the input structure and for mutating residues in the structure to nonstandard residues.

Advanced button

Set advanced options for residue scanning: calculation of pKa change, adding an implicit membrane, Monte Carlo options for affinity maturation. Opens the Advanced Options Dialog Box.

If you add an implicit membrane, you must import the structure again (by clicking Import) so that the membrane parameters are read in.

Job toolbar

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

The Job Settings button opens the MM-GBSA Residue Scanning - 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.

Tutorials

Quick Reference Sheets

Comparing Mutational Analysis Methods

The table below compares approaches for protein stability and affinity prediction for residue scanning and protein FEP+. MM-GBSA: Molecular Mechanics with Generalized Born and Surface Area Solvation. See Related Topics below for more information.

Related Topics