Running Example
In the following example, we will create a designed library of variants of a protein-protein interface (pdb code 3U43. This structures contains a complex of two proteins, one in chain A and the other in chain B. The proteins interact through an interface formed by two loops: residues 21-36 on chain A, and residues 80-100 on the chain B. Also, it is known from previous studies that residues 54A, 55A, 86B are crucial for the interaction (hotspot residues).
Note that AffiLib can also be applied to one chain in order to increase its affinity for the target chain, which stays fixed. Step 1: Calculating the sequence space
In this step you will enter the parameters of the complex to be modeled, so we may calculate the possible sequence space (i.e., the allowed amino acid identities in each interface position). Make sure you know your protein!!
Email address: coolscientist@weizmann.ac.il
Do you wish to upload your own PDB file? no
PDB ID: 3U43
Chain identifier to design: A,B
Amino acid positions to diversify: 24A, 27A, 28A, 31A, 77B, 82B, 85B, 96B, 97B
Essential amino acid residues: 54A,55A,86B
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 interface. 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 patient 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 interface mutations)
Maximal number of mutations per design: 5 (all mutants are different from the WT protein by at most 5 interface 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 R24M and P85Y are deleterious, so you remove M and Y from the list of allowed amino acids in positions 24A and 85B, 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.