Frequently Asked Questions
**If you have specific questions that were not addressed in the about page, help pages or here, feel free to write us and we will add them (

How much time takes a PROSS calculation?
The time required for PROSS to complete a calculation depends on the size of your protein and on the number of other queries running in parallel. You may receive the output email within few hours or within 1-2 days. If after 2 days you haven't received the output email, checkout the next question and if the answer is not there please contact us (

Can I submit a structure with missing density (missing residues/loops)?
Yes! You may submit a structure that has some missing residues.

Obviously, the missing residues will not be mutated, PROSS is not aware to their existence. In addition, 3 residues from each side (N and C) of the missing part will not be mutated either (because we can't be sure what is going on there from a structural point of view). In the final output however, you should get the stabilized sequences with the missing residues, but we strongly recommend that you align the final sequences with the original sequence you typically use to make sure that there wasn't any problem with the addition of the missing residues (the addition to the final sequence is based on the fasta file downloaded from RCSB or provided by the user in case of using the upload structure option).

Also note that in case you use another structure/model to define specific residue numbers to fix, make sure that the defined residues are not missing in the structure that you actually submit. Do not refer to residue numbers that are not in the submitted structure.

Can I submit a PSE file created by Pymol?
No. You can't submit a PSE file. The format of submitted structure should always be pdb.
In Pymol there is an option called "save molecule" that allows you to save specific molecules in a pdb format.

Can I submit a NMR structure?
No, you can't. Though, if you tweak the NMR-based pdb file to look as if it is based on a crystal structure (having all atoms appearing only once), you can submit. However, we strongly recommend avoiding NMR structures, they are typically not accurate enough for these kind of calculations.

Can PROSS handle non native amino acids (e.g. Selenomethionine MSE, carboxylated Lysine KCX etc.)?
PROSS turns Selenomethionines (MSE) to Methionines, Carboxlyated Lysines (KCX) to Lysines and Hydroxycesteines (CSO) to Cysteins. Other non native amino acids are recognized and spliced out. This does not affect calculations but mutations at the protein regions near the spliced amino acid (<5A) are less reliable. Consider removing them from sequences that you plan to order.
If you specify position numbers of non native amino acids that are spliced out you will get an error.

Is PROSS good for stabilizing symmetric oligomers?
No and yes. In PROSS you can design only a single chain per query. If your protein is a symmetric dimer with chains A and B for instance, you must select either chain A or B for design. If chain A was selected, chain B will be removed and PROSS "will see" a solvent at the interface region. We therefore recommend to avoid any design of chain A residues at the contact site between chains A and B. This can be easily done using the interacting chains option. In some cases (especially for higher order oligomers) using the interacting chains option will fix too many residues leaving PROSS with too little to work with. In such a case we recommend that you apply your own decisions using the manual fixation option specific residues to fix

Is PROSS good for stabilizing antibodies?
Yes, but results are probably less reliable than for other families. Specifically, mutations at CDR regions are not reliable.
If you wish to stabilize an antibody for which there isn't an available crystal structure, contact us. We may be interested in that.

Is PROSS good for stabilizing membrane proteins?
For modeling PROSS uses a standard energy function designed for soluble proteins. It does not contain any energy terms that take into account the membrane environment and in practice, it is unaware to the membrane and its location.

Is PROSS good for stabilizing small proteins (<100 amino acids)?
PROSS follows a conservative approach in order to minimize false positives. In very small proteins, you might end up with no mutations at all or with a very small number (<4) that is less likely to produce an effect. This is especially true when there are a lot of constraints (small-molecule ligands, interacting chains, specific residues were fixed). We suggest to submit free-of-constraints queries followed by removal of mutations that seem too close to active sites by eye

I submitted a query and received an error email. What should I do?
Errors are most likely due to user's wrong input.
In the error email you have a list of all the parameters that you submitted to PROSS. Go over each parameter and make sure it is correct.
Specifically, users who use the upload_pdb option and/or the upload MSA option tend to submit files that do not obey the format rules as described in the help pages.

Here is a list of common user errors that we saw in the past, listed by categories:

If you have more ideas about errors to include here let us know.

If you think that all the parameters are correct and yet the job fails, please contact us at