Dear Patrick, I don't know how many comments you got offline, but here are my comments:
1) Ice rings. Even rings that are hardly visible by eye can disturb the data such that Rfactors get stuck in the range you mention. Especially XDS, which is otherwise an excellent data processing program, performs poor in the presence of ice rings. You may want to look carefulla at your diffraction images, the wilson plot and at which resolutions you find most outlyers. 2) Crystal packing. With 2 or 4 molecules in the asymmetric unit, a~b or 2a~b and space group P(2)(1)2(1)(2)(1), you have plenty possibilities for pseudo crystallographic symmetry. Since you have high resolution data, I would process or expand the data to P1, also expand your pdb file to P1 and refine in P1 to see what happens. I would also run Phaser or some other molecular replacement program on the P1 data to see what comes out. Good luck! Herman -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll Sent: Saturday, April 27, 2013 12:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refinement hanging--what am I missing? Responding to a couple of questions from Ethan, Charlie, and Phil: Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll double-check wavelengths as a sanity check for scattering factors (but several other native data sets from the same synchrotron trip refined beautifully, so I suspect there's no gross boo-boos of this nature...) Charlie: Solvent regions are pretty clean; I haven't tried any flipping (these are molecular replacement models, so it didn't occur to me...). I tried applying NCS in one case (the smaller cell) and it had no apparent effect on the refinement. The Fo-Fc map has no strong features crying out for interpretation. Just based on geometry and map appearance, I'd be inclined to say the refinement is done, were it not for the crappy R values. Phil: I used TLS for refinement in both xtal forms; it gives a small improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply used one monomer/TLS group (these are ubiquitin variants, so the monomer itself is pretty much a little rock, without any internal domain motions). There are the usual complement of disordered side chains, but nothing unusual, and >98% of the main chain is accounted for. Haven't tried Arp/wArp yet... Excellent thoughts, keep those cards and letters coming. I'm still chewing on the substantive comments from Dean and Adrian... Thanks, Pat On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote: > Hi Pat, > > Your stats aren't all that bad, but I share your discomfort. > > Do the solvent regions retain any significant features? Have you tried > flipping those features? Have you applied NCS? What does the Fo - Fc map look > like? > > Charlie > > On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote: > >> Hi all, >> >> Here is a problem that's been annoying me, and demanding levels of thought >> all out of proportion with the importance of the project: >> >> I have two related crystal forms of the same small protein. In both cases, >> the data look quite decent, and extend beyond 2 A, but the refinement stalls >> with statistics that are just bad enough to make me deeply uncomfortable. >> However, the maps look pretty good, and there's no obvious path to push the >> refinement further. Xtriage doesn't raise any red flags, nor does running >> the data through the Yeates twinning server. >> >> Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. >> Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = >> 0.24/0.27 >> >> Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. >> Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = >> 0.21/0.26 >> >> As you would expect, the packing is essentially the same in both crystal >> forms. >> >> It's interesting to note (but is it relevant?) that the packing is quite >> dense--solvent content is only 25-30%. >> >> This kind of stalling at high R values smells like a twin problem, but it's >> not clear to me what specific kind of twinning might explain this behavior. >> >> Any thoughts about what I might be missing here? >> >> Thanks, >> >> Pat >> >> >> --------------------------------------------------------------------- >> ------------------ >> Patrick J. Loll, Ph. D. >> Professor of Biochemistry & Molecular Biology Director, Biochemistry >> Graduate Program Drexel University College of Medicine Room 10-102 >> New College Building >> 245 N. 15th St., Mailstop 497 >> Philadelphia, PA 19102-1192 USA >> >> (215) 762-7706 >> pat.l...@drexelmed.edu >
