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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: Phil Jeffrey pjeffrey {- at -} PRINCETON {- dot -} EDU Date: 2008-02-07 8 monomers), this is also the one that showed the greatest improvement in R-free once the structure was correctly redetermined (7% or 14% depending on which refinement protocols you compare). The other crystal form of MsbA and the crystal forms of EmrE didn't have particularly high-copy NCS (2 dimers, 4 monomers, dimer, 2 tetramers) and the R-frees were somewhat comparable in all cases (31-36% for the redetermined structures). The *major* source of the R-free suppression in all these cases with the inappropriate use of multi-copy refinement at low resolution. Phil Jeffrey Princeton 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 >> >> ******************************************************* >> >> > CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 07 February 2008 |
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