[ccp4bb] R/Rfree gap with pseudotranslational symmetry
Hi all, I'm having an issue during structure refinement where I can't seem to get my Rfree to drop below about 0.30 (whereas Rwork is around 0.24) and was wondering if anyone had any thoughts on the matter. I have a 2.3Å dataset that I processed the data in P31 and got an MR solution in phaser with RFZ=29.3, TFZ=14.9. There is 6-fold NCS present in the structure and it appears there is pseudotranslational symmetry with P62 as well. I have run Zanuda and P31 seems to be the correct space group. Also, Xtriage didn't detect any sort of twinning. 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. Thanks, Kim
Re: [ccp4bb] R/Rfree gap with pseudotranslational symmetry
On Fri, Sep 26, 2014 at 8:17 PM, Kimberly Stanek kas...@virginia.edu 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
Re: [ccp4bb] R/Rfree gap with pseudotranslational symmetry
Well - R / Rfree gaps are not really the thing to worry about after a MR solution. Both seem to have decreased sensibly, so now you need to worry about the map quality - can you see things to rebuild? water structure ? etc. It is possible the NC translation will affect the R factors. NC translation can make whole classes of reflections very weak, and of course weak reflections will have high R factors.. But I dont understand how an NC translation can make P31 SG be pseudo P62? ? Eleanor On 26 September 2014 21:56, Nat Echols nathaniel.ech...@gmail.com wrote: On Fri, Sep 26, 2014 at 8:17 PM, Kimberly Stanek kas...@virginia.edu 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
