Prepare has been tuned to optimally minimize the energy of a structure so that it is ready for Design. It should not require any subsequent Repack/Minimize/Relax before going into Design.
After Prepare, you can go ahead and run Design with the following number of repeats:
- 10-20 repeats for a short sequence of less than 10 residues.
- 50-100 repeats for a larger protein, or up to 200 if you are searching for more diversity in outputs.
After running Design, you may find that your sequences are all very similar or that they are fairly diverse. The Sequence Logo in Bench and the Compare tool may be helpful when analyzing the diversity of Design results.
Depending on your goal you may want diverse structures with a variety of mutations or you may want less diverse structures with a smaller variety of mutations.
If you are not getting sufficient diversity, you will need to take a more aggressive approach through any of the following options:
1.) Run Prepare again, or run it 10-50 times to get more structures. Prepare is stochastic; each output will be slightly different and will give diversity in design.
2.) Run Minimize and then Relax on a number of structures (e.g. 10-20) and then use those outputs as input for Design. Again, this will encourage diversity in designs.
3.) Most aggressively, you may perform a Relax on your structure (or many Relax runs), pick the ones that don’t drift too far (low RMSD in the filter panel) and then run Design on those. This is a fairly aggressive approach, and will give you a immense diversity, but this approach is useful if you would like to identify many different mutations.