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Re: [ccp4bb] an over refined structure |
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CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 07 February 2008Subject: Re: an over refined structure From: Axel Brunger brunger {- at -} STANFORD {- dot -} EDU Date: 2008-02-07 1. yes, even if you don't apply NCS restraints/constraints there will be correlations between reflections in cases of NCS symmetry or pseudo-crystallographic NCS symmetry. 2. Fabiola, Chapman, et al., published a very nice paper on the topic in Acta D. 62, 227-238, 2006. 3. From my experience, the effects for low NCS symmetry are usually small, except cases of pseudo-symmetry which can be easily addressed by defining the test set in the high-symmetry setting. For high NCS symmetry, the effects are more significant, but then the structure is usually much better determined, anyway, due to averaging. 4. At least the first one of the mentioned MsbA and EmrE structures had a very high Rfree in the absence of multi-copy refinement ( ~ 45%)! So, the Rfree indicated that there was a major problem. 5. The Rfree should vary relatively little among test sets (see my Acta D 49, 24-36, 1993 paper) - if there are large variations for different test set choices then the test set may be too small or there may be systematic problems with some of the reflections causing them to dominate the R factors (outliers at low resolution, for example). Axel Brunger On Feb 7, 2008, at 9:57 AM, Dean Madden wrote: > Hi Dirk, > > I disagree with your final sentence. Even if you don't apply NCS > restraints/constraints during refinement, there is a serious risk of > NCS "contaminating" your Rfree. Consider the limiting case in which > the "NCS" is produced simply by working in an artificially low > symmetry space-group (e.g. P1, when the true symmetry is P2): in > this case, putting one symmetry mate in the Rfree set, and one in > the Rwork set will guarantee that Rfree tracks Rwork. The same > effect applies to a large extent even if the NCS is not > crystallographic. > > Bottom line: thin shells are not a perfect solution, but if NCS is > present, choosing the free set randomly is *never* a better choice, > and almost always significantly worse. Together with multicopy > refinement, randomly chosen test sets were almost certainly a major > contributor to the spuriously good Rfree values associated with the > retracted MsbA and EmrE structures. > > Best wishes, > Dean > > Dirk Kostrewa wrote: >> Dear CCP4ers, >> I'm not convinced, that thin shells are sufficient: I think, in >> principle, one should omit thick shells (greater than the diameter >> of the G-function of the molecule/assembly that is used to describe >> NCS-interactions in reciprocal space), and use the inner thin layer >> of these thick shells, because only those should be completely >> independent of any working set reflections. But this would be too >> "expensive" given the low number of observed reflections that one >> usually has ... >> However, if you don't apply NCS restraints/constraints, there is no >> need for any such precautions. >> Best regards, >> Dirk. >> Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: >>> It is important when using NCS that the Rfree reflections be >>> selected is >>> distributed thin resolution shells. That way application of NCS >>> should not >>> mix Rwork and Rfree sets. Normal random selection or Rfree + NCS >>> (especially 4x or higher) will drive Rfree down unfairly. >>> >>> Doug Ohlendorf >>> >>> -----Original Message----- >>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf >>> Of >>> Eleanor Dodson >>> Sent: Tuesday, February 05, 2008 3:38 AM >>> To: CCP4BB@JISCMAIL.AC.UK >>> Subject: Re: [ccp4bb] an over refined structure >>> >>> I agree that the difference in Rwork to Rfree is quite acceptable >>> at your resolution. You cannot/ should not use Rfactors as a >>> criteria for structure correctness. >>> As Ian points out - choosing a different Rfree set of reflections >>> can change Rfree a good deal. >>> certain NCS operators can relate reflections exactly making it >>> hard to get a truly independent Free R set, and there are other >>> reasons to make it a blunt edged tool. >>> >>> The map is the best validator - are there blobs still not fitted? >>> (maybe side chains you have placed wrongly..) Are there many >>> positive or negative peaks in the difference map? How well does >>> the NCS match the 2 molecules? >>> >>> etc etc. >>> Eleanor >>> >>> George M. Sheldrick wrote: >>>> Dear Sun, >>>> >>>> If we take Ian's formula for the ratio of R(free) to R(work) from >>>> his paper Acta D56 (2000) 442-450 and make some reasonable >>>> approximations, >>>> we can reformulate it as: >>>> >>>> R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) >>>> >>>> where s is the fractional solvent content, d is the resolution, p >>>> is >>>> the effective number of parameters refined per atom after >>>> allowing for >>>> the restraints applied, d^3 means d cubed and sqrt means square >>>> root. >>>> >>>> The difficult number to estimate is p. It would be 4 for an >>>> isotropic refinement without any restraints. I guess that p=1.5 >>>> might be an appropriate value for a typical protein refinement >>>> (giving an R-factor >>>> ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R- >>>> factor ratio of 0.277/0.215 = 1.29 is well within the allowed >>>> range! >>>> >>>> However it should be added that this formula is almost a self- >>>> fulfilling prophesy. If we relax the geometric restraints we >>>> increase p, which then leads to a larger 'allowed' R-factor ratio! >>>> >>>> Best wishes, George >>>> >>>> >>>> Prof. George M. Sheldrick FRS >>>> Dept. Structural Chemistry, >>>> University of Goettingen, >>>> Tammannstr. 4, >>>> D37077 Goettingen, Germany >>>> Tel. +49-551-39-3021 or -3068 >>>> Fax. +49-551-39-2582 >>>> >>>> >>>> >> ******************************************************* >> Dirk Kostrewa >> Gene Center, A 5.07 >> Ludwig-Maximilians-University >> Feodor-Lynen-Str. 25 >> 81377 Munich >> Germany >> Phone: +49-89-2180-76845 >> Fax: +49-89-2180-76999 >> E-mail: kostrewa@lmb.uni-muenchen.de >> > >> ******************************************************* > > -- > Dean R. Madden, Ph.D. > Department of Biochemistry > Dartmouth Medical School > 7200 Vail Building > Hanover, NH 03755-3844 USA > > tel: +1 (603) 650-1164 > fax: +1 (603) 650-1128 > e-mail: dean.madden@dartmouth.edu Axel T. Brunger Investigator, Howard Hughes Medical Institute Professor of Molecular and Cellular Physiology Stanford University Web: http://atb.slac.stanford.edu Email: brunger@stanford.edu Phone: +1 650-736-1031 Fax: +1 650-745-1463 CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 07 February 2008 |
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