The Disulfidizer API runs the Rosetta “Disulfidizer” protocol. The disfulfidizer protocol attempts to stabilize a protein by making disulfide bonds. This method has been used widely with atomic-resolution success for de novo designed small proteins, and less extensively for larger proteins. The user may restrict what sets of residues the protocol searches for disulfides between.  The protocol will return protein structures for as many disulfides and combinations thereof it found to be stabilizing, as well as a scorefile list of structures.  If it is not possible to make reasonable disulfide bonds for the given backbone, no models will be returned. See Notes for more details on the disulfidizer protocol.

Quickstart

Command Line Examples

Run disulfidizer on an input structure allowing any residue to form disulfides

Run disulfidizer on an input structure in permissive mode to allow more permissive disulfide bond formation and relax output models.

Run disulfidizer on an input structure allowing residues 5-8 to form disulfides with residues 30-32

Run disulfidizer with max disulfides set to 1

Python Examples

Run disulfidizer on an input structure allowing any residue to form disulfides

Run disulfidizer on an input structure in permissive mode

Run disulfidizer on an input structure allowing residues 5-8 to form disulfides with residues 30-32

Inputs

• --pdb-file

  • Input PDB file – a cleaned and/or relaxed PDB file

  • Prepare a clean PDB using the Clean PDB API, especially when using residue sets in command.

  • Cyrus strongly recommends using the Prepare or Relax tool in Cyrus Bench to preprocess models before uploading to this API.  This will provide a more stable scoring baseline and will make interpreting results easier.

Options

• --residue-set-1 (str)

  • Space separated residue numbers which should be allowed to form disulfide bonds with the residues in --residue-set-2

  • See Notes for more details

• --residue-set-2 (str)

  • Space separated residue numbers which should be allowed to form disulfide bonds with the residues in --residue-set-1

  • See Notes for more details

• --permissive (boolean)

  • Enable more permissive disulfide bond formation instead of standard disulfide detection

  • See Notes for more details

• --max-disulfides (int)

  • Set the maximum number of disulfides that can be created per model

  • Recommended setting this value to 1 if you are modeling a larger protein

  • default = 1000

• --run-relax

  • Run Rosetta Cartesian Relax on all models output by disulfidizer

Outputs

• models (directory)

  • PDB files stabilized with the addition of disulfide bonds

  • Number of models will vary based on the number of feasible disulfide bond arrangements.

  • Models will be named in the form input_0001_####.pdb, where #### is an index (0001, increasing).  

• score.sc

  • Textfile containing Rosetta scores for each generated model

• See Notes for more information

Notes

Rosetta Disulfidizer Protocol

To summarize the documentation: 

This protocol scans a protein and builds disulfides that join residues in one set of residues with those in another set. GLY and PRO residues are ignored. Residues too close in primary sequence are ignored. Potential disulfides are first identified by CB-CB distance, then by mutating the pair to CYS, forming a disulfide, and performing energy minimization. If the energy passes a cutoff, it is compared with a set geometric criteria from a database of known disulfides in the PDB. If the “distance” along this metric passes a cutoff, the pairing is considered a valid disulfide bond.

Once valid disulfides are found, they are combinatorially added. For example, if disulfides are identified between residues 3 and 16 and also between residues 23 and 50, the following configurations will be found: 1. [3,16] 2. [23,50] 3. [3,16],[23,50]

The Rosetta Disulfidizer protocol described in further detail here (paper) and here (documentation).

Using residue sets

• This is useful for a case like the inside of a beta-sandwich protein: one set is each interior face of the protein.

• If you choose to supply residue set data, if there are already disulfides in your model, be sure that they are entirely inside or outside of your residue sets. Putting half a pre-existing disulfide in a residue set is forbidden behavior.

• Uploaded PDBs must start at residue 1 to match this numbering scheme.  If you do not intend to use residue sets, PDB numbering requirements are less strict.  

• If you supply no residue set data, the code will just look across the entire protein.  This may be contraindicated if the protein is large (you may get too many results to sort through) or if you specifically want focussed results.

• Cyrus recommends selecting residue sets, for ease of interpretation of the results.

Using permissive mode

• Cyrus has chosen best-practice settings on your behalf.  We have bundled several into one user facing option: standard versus permissive disulfide detection.

• Standard detection uses Rosetta’s default settings and will provide the most plausible disulfides.  The downside is that it will find no plausible disulfides for some protein backbones/positions.

• Permissive detection tunes the disulfide score and geometric criteria cutoffs to more permissive settings.  

  • This means that Rosetta is much more likely to find candidate disulfides.

  • Many hits will be lower quality.

    • Many hits will appear as “not disulfides” in external software such as PyMOL – they will be oriented too poorly to be shown as a proper disulfide.

  • Rosetta may find a tremendous number of disulfides (or combinations thereof) for some backbones, making the results harder to interpret.

Output File interpretation

The model numbering is irrelevant to quality. The models are ordered in a way such that the first model (input_0001_0001.pdb) will contain the most N-terminal disulfide in the set, and the last-numbered model will contain the mostly C-terminal numbered disulfides.

Model quality can be assessed via scores.  Broad documentation on interpreting scores can be found here.  The score.sc file is a space-delimited data table, padded for easier reading.  It can be parsed with pandas dataframes, excel, or your tool of choice.  In the score.sc file, pay particular attention to the total_score and dslf_fa13 columns (the second and third).  The total_score is Rosetta’s overall grade for a model.  dslf_13 is a measurement of just the quality of the disulfides in the model.  This is a total score for all disulfides, so if there are several it adds them together.  In both cases, these scores are more-negative-is-better.  The total_score should have a negative value for good models, although this will depend enormously on the quality of the input model.  The dslf_13 score should have a small positive value (single digit positive) in an ideal case.  

Disulfidizer will output all combinations of disulfides that it can (see the introduction above).  As a consequence, the disulfide score for any individual disulfide is present for the model that contains only that disulfide.  You can also find this data in the per-residue score tables at the end of each PDB file. These are also in a space-delimited tabular format that can be imported into your analytics tool of choice.

Rosetta will also report the disulfides into the PDB models in two ways:

• the standard SSBOND record

• an additional comment record

Example PDB data:

These can be parsed to get a quick overview of where disulfides went in an entire return dataset.

Treatment of existing disulfides

Disulfidizer is set up to discover disulfides even where they only exist.  As a consequence, models with existing disulfides may have them “rediscovered”, which may appear as a repacking or rotamer change.

Troubleshooting

If a job has failed because no disulfides could be formed, you will get an error message to that effect.  For any other failure contact Cyrus support for further details on the error and how to address it. 

No disulfides could be formed:

Some other error:

References

Accurate de novo design of hyperstable constrained peptides

Rosetta Disulfidize

Rosetta Energy Terms