Eva, I have used CNS to refine several low resolution structures. I have even developed several tools specifically for low-resolution refinement in CNS (see http://xray.utmb.edu/PMB/index.html#BPATCH). However, I agree with the advice given by most of my colleagues, refine isotropic B-factors in REFMAC, but do not forget to use TLS. My earlier post (see below) bounced, due to my having multiple email addresses, not all of which are privileged to post to CCP4BB. Both CNS and PHENIX have some nice features, not available in REFMAC, such as composit-omit maps and simulated annealing, which really reduce the model bias. So I often switch back and forth between CNS and REFMAC.
On Wed, 2007-04-18 at 15:51 +0200, [EMAIL PROTECTED] wrote: > Dear Eva and Harry and others, > > I am not sure that an overall B-factor is the best solution for a 3.2 > Å structure. In general, the true B-factors will vary a lot for > different parts of the protein and poor diffracting proteins often > have parts which are partially or completely disordered. An overall > B-factor is a very poor estimate of such a true B-factor distribution > and together with the low-resolution data will create a lot of model > bias i.e. very nice density for side chains which are, in fact, > disordered. > I agree, but the problem is that the traditional isotropic B-factor refinement is not restrained properly. Many years ago Ian Tickle determined that the correct restraint for isotropic B-factors had a B**2 dependence. I have continued his analysis, and have found a general restraint weight that seems to work for any resolution data. So far I have refined isotropic B-factors at 4 A, with a noticeable decrease in free-R (http://xray.utmb.edu/PMB/index.html#BPATCH), over a multiple grouped-B approach. This improved B-factor restraint patch is available with PMB (http://xray.utmb.edu/PMB), for use with CNS 1.1. Alternately, the isotropic B-factor restraints in PHENIX uses a weighted nearest-neighbor restraint which should be robust, and may possibly work at low resolution also. Another work-around is to apply very tight restraints in terms of target values. Your bond rmsds, at this resolution, should be in the 0.004 to 0.006 A range. Decreasing the B-factor restraints will help also, but will over-restrain the side-chains and short loops. Do not forget to use TLS, and I would recommend the TLSMD server (http://skuld.bmsc.washington.edu/~tlsmd/) as an easy and unbiased way to select your TLS groups. TLS can model gross B-factor variations with very few free parameters, and will generally result in a significant improvement in your model. Whichever approach you take I wish you good luck, a 3.2 A structure, in the absence of NCS, can be very difficult to build and refine. Mark On Wed, 2007-04-18 at 16:41 +0200, Eva Kirchner wrote: > Thank you Roberto, I saw that line in the log file, too. So it is as I > feared: Refmac cannot be stopped from refining B-factors ;-) Maybe > I'll use CNS... > > Eva > > > 2007/4/18, Roberto Steiner < [EMAIL PROTECTED]>: > > > On 18 Apr 2007, at 14:39, Eva Kirchner wrote: > > > > > > > > > > > > (But I'm still curious about the B-factor refinement when > > there is no "REFI BREF ISOT" in the com-file...) > > > > Eva, > > > Refmac internal default is REFI BREF ISOT > that's why even if you remove the above line from the com file > (or deselect that option from the interface) it still does > ISOT BREF refinement. > It does tell you that though.... > if you look at the log file there's a bit that says > > > ....... > Method of minimisation : Sparse Matrix > Experimental sigmas used for weighting > Number of Bins and width: 20 0.0080 > Refinement of individual isotropic Bfactors > .......... > what do you have there when you deselect the B fact option > from the interface? > > > > > I agree with Herman that at 3.2 A isotropic B values > refinement can be useful. > > > > > Roberto > > > > > > Eva > > > > > > > > 2007/4/18, Mischa Machius > > <[EMAIL PROTECTED]>: > > > > Like Harry said, it is not justified to do > > individual B factor refinement at that resolution. > > Well, you can do it, but you'll end up with funny > > results, such as what are observing right now. > > Still, from a pragmatic point of view, individual B > > factor refinement in cases like these can have a > > positive effect on the electron density. However, > > keep in mind that the resulting B factors may > > physically not be very meaningful. In the end, > > you'll have to switch to grouped B factor > > refinement, or you risk nasty comments from an > > attentive mentor or reviewer (and rightly so). Hope > > that helps. Best - MM > > > > On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote: > > > > > > > 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 > > > > > > > > > > > > > -------------------------------------------------------------------------------- > > > Mischa Machius, PhD > > Associate Professor > > UT Southwestern Medical Center at Dallas > > 5323 Harry Hines Blvd.; ND10.214A > > Dallas, TX 75390-8816; U.S.A. > > Tel: +1 214 645 6381 > > Fax: +1 214 645 6353 > > > > > > > > > > > > > > > --- > Dr. Roberto Steiner > Randall Division of Cell and Molecular Biophysics > New Hunt's House > King's College London > Guy's Campus > London, SE1 1UL > Phone +44 (0)20-7848-8216 > Fax +44 (0)20-7848-6435 > e-mail [EMAIL PROTECTED] > > > > > > > > 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
