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On Fri, Sep 26, 2014 at 8:17 PM, Kimberly Stanek <[log in to unmask]> wrote: > Before refinement in phenix the R/Rfree gap is rather small, however even > after one round of refinement I am finding that this gap increases to > almost 0.06. I have a feeling that the high symmetry present has something > to with this R/Rfree gap but was hoping some of you may have some helpful > suggestions for how to deal with it. > It's normal for the R/R-free gap to increase during the first round of refinement in molecular replacement - in fact, unless you are solving a near-identical crystal form and keeping the original R-free flags, this is almost guaranteed to happen. MR will use all reflections and the limited refinement Phaser does uses very coarse parameterization (rigid-body and group B-factor), so the R-free will usually be quite low and sometimes even lower than R-work. Restrained refinement will immediately start to open the gap, but if it's working properly, it won't keep expanding throughout refinement. At this resolution a gap in the range of 0.02-0.04 would be normal - less than this is unusual. My guess is you just need to change the relative weights of the X-ray target and geometry restraints so that the latter are stronger. Also, use NCS restraints if you aren't already. -Nat