How to use MutaBind2 Server?

Step 1 - Provide the structure of a protein-protein complex

You can either input the PDB code, the structure will be retrieved from the Protein Data Bank, or upload your own structure file, which must comply with the PDB format.

In either case, the structure file should contain at least two distinct protein chains. Please note that:

If a valid PDB code is input, the title of the PDB will be shown.

Step 2 - Define two partners for a protein-protein interaction

A user can define interaction partners by dragging and dropping (or clicking on triangles in left or right corner) any chain thumbnail into the box of first (Partner 1) or second partners (Partner 2).

Step 3 - Select mutations to make a mutant protein complex

For single mutation, one can select several mutations (amino acid substitutions) on this page but only one mutation will be introduced to the complex each time (the maximum number of single mutations that can be submitted simultaneously is 16). Please note that when more than one mutations are selected, each of them will be treated independently.

For multiple mutation, one can use “Mutation ID” to assign which mutations belong to the same multiple mutation, for instance, M1 indicates multiple mutation Number 1. The maximum number of multiple mutations is up to ten mutations, and the number of mutations that can be submitted at one time is four (M4). Similarly, Contact residues are also shown in orange in "Residue" list of "Specify One or More Mutations" and "Upload file" allows the users to submit a list of mutations on the chains specified in the second step in a plain text file.

After a user makes the selection of a chain and a position to be mutated, they can visualize the location of the mutated (substituted) residue in the wild-type complex in the 3D viewer.


Upon the completion of the third step and submission of the job, MutaBind2 provides a job identifier for checking the job status. If an e-mail address is provided, the link to the results page will be sent via e-mail. The results will be available on this page as soon as the calculations are done and they will remain on the webserver for one week. The example below shows precalculated results for job 2019061902014990892595619 and job 2019062400450556296942023 for single mutation and multiple mutation respectively.

For each protein/mutation, Mutabind2 provides the following results:

Results can be viewed directly on the browser or downloaded as a plain text file.

Homologous binding sites (only for single mutation)

MutaBind2 allows exploring homologous binding sites for each protein chain and amino acid residue that was tested. This option can be accessed by clicking on "Explore" link in the results table.

The binding sites are download from Inferred Biomolecular Interactions Server at NCBI (IBIS, It may take several seconds before the results are retrieved. IBIS server identifies protein-protein complexes and binding sites homologous to the query (protein chain that was mutated) and include the mutated site.

Note that if there is no binding site defined for the protein chain, or if the mutated residue is not part of any binding site in IBIS no results will be displayed. Interactions with different partners are shown separately; see IBIS for partner description. In some cases, as in the example above, IBIS does not identify any homologs, thus only the binding site for query protein is shown. Otherwise, see another example below (open precalculated job), the table will show alignments of binding site residues colored according to their conservation (red denoting highly conserved positions). Mutated site is highlighted in yellow. One can click on a residue in a homologous structure to check it in MutaBind2, which will require submitting a new job for calculation.

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