Eva, If you do have NCS then you should be aware of a bug in the CNS1.1 release. PMB has a patch to fix this, and I hope that CNS1.2 will have fixed it also. The problem is with the NCS restraints on B-factors in CNS. The NCS groups, usually molecules, are restrained to have identical B-factors. This does not allow for the affect of local environment on the motions of the separate molecules. The PMB patch basically applies an overall B-factor to each NCS-related group to account for differences in the local environment of each group, such as differing crystal contacts. The effect is sometimes quite significant (B-factors ~10), but on other occasions the overall B-factors can be ~0, indicating that the molecules are practically identical.
Good Luck, Mark On Thu, 2007-04-19 at 10:07 +0200, Eva Kirchner wrote: > Hi all, > > thanks for all your help so far, and as we ended up in a more general > discussion about temperature factor refinement at not-so-great > resolution, here is a quick summary of what I'll try out: > > 1.) Refine overall B's instead of isotropic B's. > > 2.) Use isotropic B's with the following (combinable) options: > > a) Isotropic in the beginning, grouped B's in the end. > b) Use tight geometric restraints (I'm doing this anyways). > c) Use tight B restraints rather than grouped CNS B's (not > geometrically restrained, and most likely not restrained by NCS). > > 3.) Use not so tight B restraints, but tight geometric restraints with > individual or grouped B's, plus TLSMD server and multi-group TLS > refinement. > > 4.) Use CNS and try Mark White's tools, and simulated annealing. > > 5.) Use phenix with weighted nearest-neighbor restraint. > > ...and some remarks: > * Of course I never wanted not to refine any B factors! I just wanted > to see their contribution/influence on the refinement and explain > their strange behaviour. > * Luckily, I have NCS :o) Thanks for your good wishes, Mark. > > > Eva > > > > > > 2007/4/18, Eva Kirchner <[EMAIL PROTECTED]>: > > Hi, > > I have a little problem with B-factor refinement. I'm using > the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A > (I tried 8-3.2 A as well, it doesn't make a big difference for > this problem), and a current Rfree of 30.4%. > > Refmac refines the B-factors so that they are nearly the same > for main chain and side chain, and I don't like that (or could > it make sense in any way?). Moreover, my structure is a > protein complex, and Refmac is mainly doing this for one > component of the complex. If I take the B-factors from the > original uncomplexed protein (around 18, 1.75 A) and add 44 to > them with moleman to get them in the range they are in the > complex, Refmac "flattens" them remarkably in only 5 cycles of > restricted refinement. Does anyone have an explanation for > this? I am pretty sure that the complex components are in the > right place, I see beautiful density and everything I should > see at this resolution. > > Here is what I tried further: > > * I de-selected "Refine isotropic temperature factors" in the > Refmac interface. There was no REFI BREF ISOT any more in the > com file. But there was also no difference in the B-factors > compared to when there _was_ REFI BREF ISOT in the com file... > So does Refmac just _ignore_ my wish not to refine B-factors? > (The REFI keywords were as follows: type REST - resi MLKF - > meth CGMAT - is there any B-factor-thing hidden in this?) > > * I played around with the geometric parameters. If I select > the B-factor values there (the keywords are TEMP|BFAC > <wbskal><sigb1><sigb2><sigb3><sigb4>), it does not make _any_ > difference, what values I fill in there, the resulting > B-factors are always the same (but different from when I don't > use the TEMP keyword, and even "flatter"). Default for WBSCAL > is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers > for the sigbs. > > Thanks for any thoughts on this, > > Eva Sincerely yours, Mark A. White, Ph.D. Assistant Professor, Dept. Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Fax. (409) 747-4745 mailto://[EMAIL PROTECTED] http://xray.utmb.edu http://xray.utmb.edu/~white
