For most CAD modeling tools, a single run will be insufficient at sampling the variety of protein conformations. Each run begins with its own unique trajectory and one run will have a slightly different answer from another run.
The more possible conformations your protein may have, for example if it is especially flexible or large, the more repeats will be required in order to fully sample the possibilities. Additionally if you believe that your final structure will be significantly different than your starting structure, more repeats will be required.
For most cases, 100 repeats will be more than sufficient. Once you have completed a run with 100 repeats you can check whether 100 repeats is sufficient by looking at the at the range of structures’ scores, which corresponds to the energy of the structure. For an easier view of the range of structures’ scores use the Scatter Plot to view the scores of all 100 structures.
- If the range of scores is greater than 50 Rosetta Energy Units (REUs), use the lowest energy structure from that run as the input for another run this time with 200 repeats.
- If the energy range is greater than 100 or 200 REUs, use the lowest score structure as the input for another run with 500 or 1,000 repeats respectively.
Once you have fully sampled the search space, the scores for the structures’ will stop improving. Continue repeating iterative rounds of repeats as detailed above to reach this point.
Reasons to increase the number of repeats are case-specific…
- Repack – Samples side-chain orientations. Increase repeats for large, flexible proteins.
- Minimize – Only 1 run is necessary because this method differs than other sampling method. It is deterministic and will only find one answer no matter how many times you run it.
- Relax – Samples global structural changes. Increase repeats for large, flexible proteins.
- Loop Rebuild – Samples loop structure. Upper limit is 12 residues. Increase repeats when running Aggressive mode.
PROTEIN DESIGN METHODS:
- Design – Samples side chain orientation and allows mutation.
- Flex Design – Samples global side chain orientation, samples backbone near area of design.
- Relax Design – Samples global side chain and backbone and allows mutation.
- Increase repeats for large, flexible proteins.
- Increase repeats when more permutations are possible.
- Allow more repeats for Flex Design than Design. And more repeats for Relax Design than Flex Design.
HOW TO CALCULATE NUMBER OF PERMUTATIONS:
Example – For 5 mutations position that are all allowed to sample all 20 amino acids,
- The number of permutations = 20 x 20 x 20 x 20 x 20 = 3.2 Million.
- Number or repeats needed = # of permutations / 50 = 64k.
It is not recommended to run that many repeats, so we recommend splitting the job into 2 runs:
- The first run has 20 x 20 = 400 permutations, 8 repeats.
- The second run has 20 x 20 x 20 = 8,000 permutations, 160 repeats.
- Then use the preferred mutations from each run to make a final design at all 5 positions