Running Example
In the following example, we will create an active site variant library in the protein with the pdb code 5JFM. The protein was crystallized with multiple chains, but we choose to model the variants on chain A. The active site residues are: 199, 329, 331, 472, 480, 483, 493, 495. Also, it is known that residue 330 is critical for catalysis.
It is highly recommended to work on a PROSS stabillized variant of your protein.

Step 1: Calculating the sequence space
In this step you will enter the parameters of your protein to be modeled, so we could calculate the possible sequence space (i.e., the allowed amino acid identities in each active site position). Make sure you know your protein!!
Email address:
Do you wish to upload your own PDB file? no
Chain identifier to design: A
Amino acid positions to diversify: 199A,329A,331A,472A,480A,483A,493A,495A
Essential amino acid residues: 330A
Ligand designation: leave empty in this case
Use default parameters for the multiple sequence alignment generation.

Step 1.5: Wait for the first step calculation results
After setting the parameters for the first step, we calculate the possible sequence space in the active site. It might take a few hours/ days.
When the first step is done, you will receive an email with a link to select the parameters for the second step (below).

Step 2: Modeling all the multipoint mutants from the sequence space
The parameters set in this step will restrict the exhaustive modeling of all possible multipoint mutants.
Important: be patiant when clicking verify. Some calculations are long and sometimes the page loads very slowly. We are working to make response times faster!
Minimal number of mutations per design: 2 (all mutants are different from the WT protein by at least 2 active site mutations)
Maximal number of mutations per design: 5 (all mutants are different from the WT protein by at most 5 active site mutations)
Minimal PSSM threshold: 0 (this threshold value- zero, is pretty high, so the sequence space will include only amino acids that are slightly favored by the multiple sequence alignment)
ΔΔG: 1 (increase this parameter if the Total number of designs is low)
Change the values of the different parameters and click verify to check whether this parameters combination is acceptable (at least 500 mutants, and not more than 500,000) until you get a high enough number of mutations.
When satisfied with the resulted sequence space, press the proceed button to proceed to a screen where you will be able to edit manually(add and/ or remove) the sequence space. For example, you know that K155R and F495Y are deleterious, so you remove R and Y from the list of allowed amino acids in positions 155 and 495, respectively.

Step 3: Results
A few hours/ days after you submit the second step parameters you will get an email with the results. Please read carefully the attached ReadMe.txt file!!
See the FAQ for furthur details.