On Thu, Oct 2, 2014 at 7:34 AM, Sneha Rangarajan <rsneha@umd.edu> wrote:

1) When is a good time to start building the peptide into the density (should I wait until Rfactors improve significantly)?

I would advise waiting until the rest of the model is as good as possible. That way you can use the difference map as a paper figure without needing to do any extra refinement to remove model bias later.

2) If so, should I be masking the peptide density somehow so that further refinement doesn't flatten out that density?

Further refinement is probably going to make the density clearer, if it's at full (or near-full) occupancy.

3) What is a general good strategy for lowering Rfactors? (Esp if the gap between Rwork and Rfree is high..like 28/40 or so)?

Several things to try:

- much longer refinement, say 10 macro-cycles instead of 3 (and even more may be helpful)

- simulated annealing - although this sometimes has a tendency to overfit, but right now that's not as big a problem

- weight optimization will reduce the gap between R-free and R-work, although it will also slow down convergence

But you should also try to make the model as complete as possible - AutoBuild usually leaves out some residues, and rebuilding them should reduce your R-factors by quite a bit. You may be able to simply copy over appropriate pieces of the original model if they fit the map well (or can be adjusted easily).

-Nat