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Re: [ccp4bb] elusive DNA density |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: elusive DNA density From: Jacob Corn jcorn {- at -} BERKELEY {- dot -} EDU Date: 2007-11-07 While working with a low-affinity, nonspecific DNA binding protein, I ran into this problem dishearteningly often. In several cases, even when running washed crystals out on a gel had told me that they contained both DNA and protein (32P-labeling for DNA, Coomassie for protein), I was unable to obtain convincing difference density for nucleic acid. As others have suggested, this could be caused by either low occupancy or multiple binding registers in the same crystal. One way to overcome the affinity issue might be backsoaking the crystals into a lower salt concentration, though this method was never successful for me. You could also continue your screening efforts and cross your fingers for a crystal form where the DNA is better ordered. In the end, I developed a screen ("FASTDXL") based on Greg Verdine's protein-DNA disulfide crosslinking chemistry to tether the nucleic acid outside the binding site, thereby greatly increasing the local concentration and fixing DNA in a unique register (Structure 2007, 15(7):773-80; NSMB in press). If you decide to pursue something along those lines and have success obtaining a structure, be sure to rigorously validate its biological relevance. Feel free to send me an email if you have any questions about FASTDXL. Jacob Jacob Corn The Berger Lab UC Berkeley - Molecular and Cell Biology jcorn@uclink.berkeley.edu phone: 510-643-9491 fax: 510-643-9290 Melody Lin wrote: > Dear all, > > I've been working on a series of DNA-protein complex structures. In my > recently acquired data sets, I got almost no density for DNA if I do > molecular replacement or rigid body fitting with the protein structure, > although I am 100% sure I have DNA in the structure by indepenent means. > If I use models with DNA, I could find some DNA density with those data > sets, but as I refine the structure, the density became very poor. The > resolutions for those data sets are between 2.0-2.4 A. Also, if I use > the scaled data from synchrotron rather than the re-scaled data at home, > I got better DNA density, although for re-scaling, I used site > parameters that I copied done from synchrotron. The only differences > between those two sets of scaled data are: (1) the original scaled data > take into account all reflections, including high resolution data with > low completeness/redundancy, which are cut in the re-scaling; (2) error > models were changed so chi squares for each bin are 0.8-1.2 for re-scaling. > > My (very naive) questions are: (1) Does the DNA density I saw in the > cases where I use models with DNA for MR/rigid body fitting only reflect > model bias? (2) are simulated annealing or cycles of coordinate/B factor > refinement enough to get rid of model bias? (3) Does weak DNA density > have to do with data processing? > > Thanks very much for any suggestion, > Melody Lin CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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