| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | | |
|
|
Re: [ccp4bb] an over refined structure |
|
CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 08 February 2008Subject: Re: an over refined structure From: Dirk Kostrewa kostrewa {- at -} LMB {- dot -} UNI-MUENCHEN {- dot -} DE Date: 2008-02-08 Peter Zwart gave me a similar reply. This is very interesting discussion, and I would like to have a somewhat closer look to this to maybe make things a little bit clearer (please, excuse the general explanations - this might be interesting for beginners as well): 1). Ccrystallographic symmetry can be applied to the whole crystal and results in symmetry-equivalent intensities in reciprocal space. If you refine your model in a lower space group, there will be reflections in the test-set that are symmetry-equivalent in the higher space group to reflections in the working set. If you refine the (symmetry-equivalent) copies in your crystal independently, they will diverge due to resolution and data quality, and R-work and R- free will diverge to some extend due to this. If you force the copies to be identical, the R-work & R-free will still be different due to observational errors. In both cases, however, the R-free will be very close to the R-work. 2). In case of NCS, the continuous molecular transform will reflect this internal symmetry, but because it is only a local symmetry, the observed reflections sample the continuous transform at different points and their corresponding intensities are generally different. It might, however, happen that a test-set reflection comes _very_ close in reciprocal space to a "NCS-related" working-set reflection, and in such a case their intensities will be very similar and this will make the R-free closer to the R-work. If you do not apply NCS- averaging in form of restraints/constraints, these accidentally close reflections will be the only cases where R-free might be too close to R-work. If you apply NCS-averaging, then in real space you multiply the electron density with a mask and average the NCS-related copies within this mask at all NCS-related positions. In reciprocal space, you then convolute the Fourier-transform of that mask with your observed intensities in all NCS-related positions. This will force to make test-set reflections more similar to NCS-related working-set reflections and thus the R-free will be heavily based towards R-work. The range of this influence in reciprocal space can be approximated by replacing the mask with a sphere and calculate the Fourier- transform of this sphere. This will give the so-called G-function, whose radius of the first zero-value determines its radius of influence in reciprocal space. To summarize: (i) One can't directly compare crystallographic and non- crystallographic symmetry (ii) In case of NCS, I have to admit, that even if you do not apply NCS-restraints/constraints, there will be some effect on the R-free by chance. So, my original statement was too strict in this respect. But only if you really apply NCS-restraints/constraints, you force to bias the R-free towards the R-work with an approximte radius of the G- function in reciprocal space. What an interesting discussion! Best regards, Dirk. Am 07.02.2008 um 18:57 schrieb Dean Madden: > 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 >> 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 ******************************************************* 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 <-- 08 February 2008 |
| ProteinCrystallography.org: Copyright 2006-2008 by Quid United Ltd |