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On 12/21/14, 7:00 PM, "CCP4BB automatic digest system" <lists...@jiscmail.ac.uk> wrote: > There are 2 messages totaling 244 lines in this issue. > > Topics of the day: > > 1. Cross-validation when test set is miniscule > 2. non-specific disulfide bonds in crystal structures > > ---------------------------------------------------------------------- > > Date: Sat, 20 Dec 2014 17:41:00 -0800 > From: Axel Brunger <brun...@stanford.edu> > Subject: Re: Cross-validation when test set is miniscule > > Dear Derek, > > I suggest you try 10% for the test set. You should still be able to judge the > effect of > various restraints (or constraints) as long as you keep the same test set. If > you switch test sets, and re-refine, Rfree > might change as much as 2% for a test set consisting of 200 reflections - see > Fig. 6 in ref. (A. T. Brunger, Free > R value: Cross-validation in crystallography, Methods in Enzym. 277, 366-396, > 1997). However, using the > same test set may allow you to judge the best restraints protocol or weights. > > Axel > > PS: The Methods in Enzym. review also briefly discusses "complete > cross-validation". > > PPS: For refinement at very low resolution, see also: > > A.T.Brunger, P.D.Adams, P.Fromme, R.Fromme, M.Levitt, G.F. Schroder. Improving > the accuracy of > macromolecular structure refinement at 7 A resolution. Structure 20, 957-966 > (2012). > > >> On Dec 20, 2014, at 1:05 AM, CCP4BB automatic digest system >> <lists...@jiscmail.ac.uk> wrote: >> >> Date: Fri, 19 Dec 2014 11:18:37 +0000 >> From: Derek Logan <derek.lo...@biochemistry.lu.se> >> Subject: Cross-validation when test set is miniscule >> >> Hi everyone, >> >> Right now we have one of those very difficult Rfree situations where it's >> impossible to generate a single meaningful Rfree set. Since we're in a bit of >> a hurry with this structure it would be good if someone could point me in the >> right direction. We have crystals with 1542 non-H atoms in the asymmetric >> unit that diffract to only 3.6 Å in P65, which gives us a whopping 2300 >> reflections in total. 5% of this is only about 100 reflections. Luckily the >> protein is only a single point mutation of a wild type that has been solved >> to much better resolution, so we know what it should look like and I simply >> want to investigate the effect of different levels of conservatism in the >> refinement, e.g. NCS in xyz and B, group B-factors, reference model, >> Ramachandran restraints etc. However since the quality criterion for this is >> Rfree I'm not able to do this. >> >> I believe the correct approach is k-fold statistical cross-validation, but >> can someone remind me of the correct way to do this? I've done a bit of >> Googling without finding anything very helpful. >> >> Thanks >> Derek >> ________________________________________________________________________ >> Derek Logan tel: +46 46 222 1443 >> Associate Professor mob: +46 76 8585 707 >> Dept. of Biochemistry and Structural Biology >> www.cmps.lu.se<http://www.cmps.lu.se> >> Centre for Molecular Protein Science www.maxlab.lu.se/crystal >> Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com > > Axel T. Brunger > Investigator, Howard Hughes Medical Institute > Professor and Chair, Dept. of Molecular and Cellular Physiology > Stanford University > > Web: http://atbweb.stanford.edu > Email: brun...@stanford.edu > Phone: +1 650-736-1031 > > ------------------------------ > > Date: Sun, 21 Dec 2014 10:14:00 +0100 > From: mesters <mest...@biochem.uni-luebeck.de> > Subject: Re: non-specific disulfide bonds in crystal structures > > Hello Todd, > > if your protein is a cytosolic protein (reductive environment; for ER, > Golgi, mitochondria things look different), the disulfide bond formation > you observe is most probably the result of the oxidative environment > (outside the cell) in combination with the crystal packing that that > brings the two cysteins in close proximity. > > J. > > > Am 20.12.14 um 18:52 schrieb Todd Jason Green: >> Hello All- >> >> I have recently determined a domain structure of a larger protein. The >> structure shows a clear disulfide bond between two monomers in the >> asymmetric unit. I'm trying to figure out if this is an artifact of >> the crystal packing or has biological relevance. The protein has been >> reported to function as a monomer. If I look at the pool of protein on >> a SDS-PAGE gel under non-reducing conditions, I see that a smaller >> percentage (~15-20%) of the protein runs as a dimer. In the structure, >> the association has 2-fold symmetry with about 29% of the monomeric >> surface area buried between the dimer. Can anyone point me in the >> direction of a paper describing a non-specific disulfide in a crystal, >> or perhaps a criteria for assessing specificity? I will do some >> functional studies, but I'm looking for some info on a lazy saturday. >> >> Thanks in advance. Best- >> Todd >
