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[ccp4bb] Old High B-Factor Issue |
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CCP4bb navigationCCP4bb <-- 2007 <-- June 2007 <-- 09 June 2007Subject: Old High B-Factor Issue From: Gregg Crichlow gregg {- dot -} crichlow {- at -} YALE {- dot -} EDU Date: 2007-06-09 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 |
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