Hi Patrick, Did you try using a different refinement program (e.g. Refmac)? Which type of NCS restraints did you use, global or local (torsion- or distance-based)? Have you tried optimizing your restraint weights? Have you tried running a huge number of refinement cycles? You can also try running PDB_REDO (plug plug) which will try a number of things to improve your model.
Cheers, Robbie > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Patrick Loll > Sent: Saturday, April 27, 2013 00:32 > 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 > >
