Cysteine Mutation Panel

This panel allows you to find residues that can be mutated to Cys and form disulfide bonds, and to mutate Cys to other residues to break disulfide bonds.

To open this panel, click the Tasks button and browse to Biologics → Cysteine Mutation.

Using
Features
Additional Resources

Using the Cysteine Mutation Panel

Disulfide bridges between cysteine residues add to the stability of a protein structure. Mutating residues to form or break disulfide bridges offers a way of controlling the stability of a protein.

The Cysteine Mutation panel can analyze a single protein for potential mutations, such as a structure from the PDB, or it can analyze a molecular dynamics trajectory, to find frames in which suitable residues come close enough to form disulfide bridges.

The basic workflow for using this panel is as follows:

Display the protein you want to analyze in the Workspace. You should ensure that it is properly prepared (with the Protein Preparation Workflow Panel).
If you want to analyze only part of the protein, select the residues that you want to analyze, and select Analyze only selected Workspace residues.
Click Analyze Workspace or Analyze MD Trajectory

When the analysis finishes, the Residue pairs for mutation table is populated with the residue pairs that were found.
Filter the list to show only the mutations you are interested in:
- Use the display options to display the mutation types that you are interested in.
- Use the menus below the table to select the residues that you want to mutate to or from. The table is updated to show only those residues.
- Use the cutoffs to filter out pairs that do not meet specified criteria.
Select the pairs that you want to mutate. Each pair is mutated independently to produce a structure and an energy evaluation.
Decide whether you want to optimize residues near to the mutated residues, by selecting from the Minimization shell options, and choose a minimization method. The mutated residues are always optimized, by sampling the side-chain conformations to find the best geometry, then minimizing to relieve strain.
Click the Settings button,

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.
When the job finishes, go to the Results tab. If the results are not listed in the Mutations table, click Load Results from Previous Run, navigate to and open the output Maestro file from the job.
Click a row in the Mutations table to show the mutated residues in the Workspace, rendered in ball-and-stick. If you want to see the original residues as well, select Display original structure in gray.

The output structures are included in the project, so you can use them for other purposes. Depending on how many residues you included in the optimization around the mutated pair, you might want to minimize the structure with fewer constraints before using it in another application.

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

$SCHRODINGER/run cysteine_mutation_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 cysteine_mutation_backend.py Command Help.

Run tab

Analyze Workspace button

Click this button to analyze the Workspace, to locate possible mutation sites for forming or breaking Cys-Cys disulfide bonds. When the analysis is done, the residue pairs are listed in the Residue pairs for mutation table.

Analyze only selected Workspace residues option

Analyze only the residues that are selected in the Workspace to locate possible mutations. By default, the entire Workspace structure is analyzed.

Analyze MD Trajectory button

Click this button to analyze an MD trajectory to locate possible mutation sites for forming Cys-Cys disulfide bonds. The entire trajectory is analyzed to locate frames in which residues come close enough to form disulfide bridges. Opens a file selector, in which you can locate a Desmond MD simulation results file (-out.cms). After selecting the file, you are prompted to specify the interval at which the analysis is performed on the trajectory, as a number of steps. The analysis takes some time, and is run under job control. When the analysis is done, the residue pairs are listed in the Residue pairs for mutation table.

Residue pairs for mutation table

This table lists the pairs of residues that have the potential to form or break disulfide bonds. The table rows that are shown are controlled by the options below the table. The cysteine mutation job mutates only the pairs that are selected in the table, so you must make a selection before you run the job. Each selected pair is mutated independently: there are no simultaneous mutations.

The table columns are as follows:

Residue pair index

The first column contains the index of the residue pair. When the table is filtered to show only certain residue pairs, this index remains the same (i.e. it is not a table row number).

Type

Mutation type, which can be one of the following:

X-X -> S-S	Mutation of two residues to cysteine with formation of a disulfide bond.
S-X -> S-S	Mutation of one residue to cysteine with formation of a disulfide bond to a nearby cysteine.
S-S -> S-X	Mutation of one cysteine residue of a pair to break a disulfide bond.

Residue 1

Identity of the first residue in the pair, given by the chain letter, the residue number and insertion code, and the 3-letter residue name. For Cys-Cys pairs, this residue is the residue that is mutated, so the pair is listed twice, in opposite order, to allow selection of only one of the pair to mutate.

Residue 2

Identity of the second residue in the pair, given by the chain letter, the residue number and insertion code, and the 3-letter residue name.

β-carbon Distance

Distance in angstroms between the beta carbons (CB) of the two residues. In the case of Gly, the distance is taken from the alpha hydrogen (HA) instead.

Separation

Sequence separation between the residues in the pair, defined as the number of residues between the two residues. Displayed as N/A if the residues are in different chains.

SASA

Combined solvent-accessible surface area of the two residues in the pair.

Sec. Structure

Secondary structure elements of the two residues in the pair (helix, strand, etc.). If both have the same secondary structure element, it is only given once, otherwise the two elements are given in the form element1/element2.

B Factor

Temperature factors (crystallographic B factor) of the two residues in the pair, represented as B(residue 1)/B(residue 2).

Frame

Trajectory frame, if an analysis was performed on a trajectory. Each pair can come from a different frame, and the structure in that frame is the one that is mutated.

Filter menu

Filter the interactions shown in the table. Select the options from the desired Interaction type:

Cys-Cys—Show only cysteine-cysteine pairs, for S-S -> S-X mutations. The first residue listed is the one mutated, so a given pair is listed twice, in opposite order, to allow selection of either residue of the pair for mutation.
Cys-X—Show only pairs with one cysteine, for S-X -> S-S mutations.
X-X—Show only pairs of non-cysteine residues, for X-X -> S-S mutations.

To select interactions between chains, select specific chains for set A and set B from the Interaction between chains menu.

Fit on select option

Zoom to the selected residue pairs in the Workspace when a pair is selected.

X->Cys replacement residues option menu

Choose the residues that you want to consider for replacement with cysteine from this option menu. The choice changes the residue pairs that are listed in the table. The option menu contains individual residues, which you can select independently, an All option to select all residues (except Pro), a None option to clear the list, and a Conservative option to select conserved residues (GLY, ILE, LEU, VAL, ALA). The residues that you choose are displayed in the main part of the option menu; the complete list is shown in a tooltip if it is too long. The table is updated for each selection that you make.

Minimum sequence separation between pairs text box

Specify the minimum sequence separation allowed between pairs of residues in the same chain. The separation is the number of residues between the residues in the pair.

Beta carbon distance cutoff text box

Specify the maximum Cβ–Cβ allowed distance between the beta carbons of the residues in a pair. This distance is used to filter the table to show only residues with a smaller distance. For Gly, the distance is taken from the alpha hydrogen.

Minimum B factor text box

Specify the minimum B factor that at least one residue in the pair must have.

Allow only pairs where option and menus

Specify secondary structure element requirements for one or both of the residues in the pair, by selection from the menus. The requirement can include or exclude secondary structure elements.

Solvent accessible surface area option, menu, and text box

Specify the minimum or maximum combined solvent-accessible surface area for the residue pair. Note: when looking for residues that are not solvent-exposed, we recommend a SASA cutoff value greater than 80 to 100 Angstroms, instead of the default cutoff value of less than 100 Angstroms.

Cys->X replacement residues option menu

Choose the residues that you want to consider for replacement of cysteine from this option menu. The choice changes the residue pairs that are listed in the table. The option menu contains individual residues, which you can select independently, an All option to select all residues (except Pro), a None option to clear the list, and a Conservative option to select conserved residues (GLY, ILE, LEU, VAL, ALA). The residues that you choose are displayed in the main part of the option menu; the complete list is shown in a tooltip if it is too long. The table is updated for each selection that you make.

Minimization shell options

These options allow you to choose how much of the protein around the two residues involved in the mutation are optimized. The choices are:

Adjacent residues in sequence—Optimize the N residues next in the sequence on either side of the residues in the pair, where N is selected from the option menu.
None—Do not optimize any residues but the two residues in the pair.
Residues within N Å—Optimize all residues that have atoms within the specified distance of the mutated residue pair.

Refinement option menu

Choose the refinement method from this option menu, from Gas phase minimization, Implicit solvent minimization, or None.

If the solvent-accessible surface area of the minimization region is negligible, the implicit solvent model may not be of any value; if the SASA is large enough, the implicit solvent model should probably be used.

Results tab

Load Results from Previous Run button

Load the results of a previous cysteine mutation job. Opens a file selector, in which you can browse to the output Maestro file (-out.maegz).

Mutations table

This table displays the results of the mutation job. Selecting a row in the table zooms in on the mutated pair in the Workspace. The mutated residues are displayed in ball-and-stick, and the rest of the structure uses a darker color scheme. The table columns are described below.

Residues	Chain name, residue number and insertion code of both residues in the mutated pair.
Original	3-letter names of original residues in the pair.
Mutated	3-letter names of mutated residues.
Δ Ei	Change in interaction energy between the residue pair and the rest of the protein on mutation. A negative value favors the mutation.
Δ Strain E	Change in strain energy on mutation. The strain energy is the difference in internal energy between the state of the residue pair in the protein and the relaxed state of each residue or of the disulfide in the gas phase. A negative value favors the mutation.
Δ Ei+Strain E	Change in the sum of interaction energy and strain energy on mutation, equal to Δ Ei + Δ Strain E. A negative value favors the mutation.
Pre-min Score	Geometric energy score of the mutated protein prior to minimization. The score is calculated using an empirical function that is derived from the distributions of the internal coordinates of cysteine disulfides in the PDB. Geometries that are seen in the PDB for disulfides yield lower scores. Lower scores are more favorable.
Weighted Score	Weighted sum of change in interaction energy, change in strain energy, pre-minimization score and post-minimization score. The last of these uses the same method as the pre-minimization score and is reported in the Project Table, but is not reported here. The score includes a shift for mutations for which any of the four components of the weighted score is larger than a threshold for that component. Sorting the weighted score places the mutations that pass all threshold tests first, followed by all the others. Lower scores are more favorable.

Display original structure in gray option

In addition to the mutated residue pair, show the original residue pair in gray rather than colored by element.

Fit on select option

Zoom to the selected mutated residue pairs in the Workspace when a pair is selected.

Export button

Export the results in the table to a CSV file. Opens a file selector, in which you can navigate to a location and name the file.

Job toolbar

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

The Job Settings button opens the Cysteine Mutation - 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

Disulfide Bond Engineering