| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | | |
|
|
Re: [ccp4bb] Old High B-Factor Issue |
|
CCP4bb navigationCCP4bb <-- 2007 <-- June 2007 <-- 09 June 2007Subject: Re: Old High B-Factor Issue From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV Date: 2007-06-09 thing to do, especially when you realise that detwinning data with twin fraction larger than 45% is done using model information in CNS: the detwinned data you get out is not experimental anymore. Subsequently, you cannot trust your R and Rfree statistics as you would normally and any 'improvement' you see in your free R or your maps, might be due to the 'detwinning' Have you tried using TLS refinement in combination with twin refinement? Peter 2007/6/9, Gregg Crichlow > > > > > Dear CCP4 Readers, > > Quite some time ago, before the CCP4BB changed its server, I > posted the following inquiry concerning high B-factors in a protein-DNA > complex from a twinned crystal in which most of the DNA was not visible. > > > > Dear CCP4BB: > > We have a protein-DNA complex from a twinned crystal (space group P31). I > detwinned the data and refined the structure to 2.1 A with Rfree=28.8% > before adding water and nucleotides. We can only observe density for two of > the 25 nucleotides. I noticed that the B-factors are very high (around 50-60 > A^2). Not many water molecules can be added using a 80 A^2 cutoff. We > determined the structure of the unbound protein, and the B-factors are > almost as high. I tried fixing the B-factors at 20, but Rfree increased to > 30.9%. Is this indicative of an error that can be fixed? Thank you. > > > > I do apologize for the delay in the follow-up. I thank everyone who > responded. The real problem was not the B-factors, of course, but the > incomplete DNA density. The various replies I received in essence mentioned > that (1) high B-factors are not necessarily a problem, (2) I should not > refine against de-twinned data, and (3) try density modification to be able > to see more of the structure. Unfortunately, the density modification > suggestion did not work with respect to improving the map, and refining > against twinned data had not appeared to be useful. It turned out that > de-twinning the data was the best way to proceed, judging by R/R-free > statistics, although this was not supposed to be as good as working with the > twinned data using the twinning fraction. I remember noting in the CNS > tutorial that using twinned data may not work well if the twinning fraction > is greater than 0.4, which it was in our case (about 0.49). Maybe that was > the problem. In the end, I used the data de-twinned in CNS as if it were a > perfect twin. Nonetheless, the high B-factors seem to be real. Although the > Wilson B-factor is high, maybe I was just hoping that this was due to the > DNA, not so much protein, and that sharpening the protein B-factors would > improve the map. This did not work. However, after handling the data the way > we did and now obtaining reasonable statistics, at least now I can be more > confident that the electron density for the nucleotides that we do see are > real. I thank everyone for their input. > > > > Gregg > > > > > > ******************************************* > Gregg Crichlow > Dept. of Pharmacology > Yale University > P.O. Box 208066 > New Haven, CT 06520-8066 > ******************************************* > > CCP4bb navigationCCP4bb <-- 2007 <-- June 2007 <-- 09 June 2007 |
| ProteinCrystallography.org: Copyright 2006-2008 by Quid United Ltd |