I sent this on Friday to Gianluca but forgot to also send it to the bulletin board, so I’m posting it now, as it relates to Eleanor’s response.
__________________________________________________ It would also be worthwhile to consider the possibility of out-of-register errors in the sequence assignment for your model. I have seen cases at a similar resolution limit and anisotropic that had high free-R values due to this problem. A sequence assignment error in 10-15% of the the total scattering mass can be the difference between free R-values of 38% versus 32%, for example. With low resolution models, one frequently has one or two (or more) of what I like to refer to as “disembodied helixes”—I.e., helixes that have both N- and C- termini with insufficient electron density to connect them to the rest of the protein. These are great candidates for out-of-register errors. I find Buccaneer to be a great program for re-building an initial MR model and correcting such errors. It won’t fix everything, especially if the local electron density is poor, but it does a surprisingly good job with maps in the 2.8-3.6 Angstrom range. And if you’re reasonably sure of the sequence assignment in the majority of the structure solution, you can fix the portion that you think is correct and ask it to re-build the questionable part. It’s my go-to method for these type of problematic structures, Diana ************************************************** Diana R. Tomchick Professor Departments of Biophysics and Biochemistry University of Texas Southwestern Medical Center 5323 Harry Hines Blvd. Rm. ND10.214A Dallas, TX 75390-8816 diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu> (214) 645-6383 (phone) (214) 645-6353 (fax) On Oct 14, 2017, at 9:10 AM, Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote: I would be worried by a structure with these R values, whether or not there is anisotropy to consider. Several times when this has happened to me the spacegroup turns out to be wrong. You do not give any details of the solution but especially if there is some non-crystallographic translation you can assign the space group wrongly - get a MR solution which fits some of the amplitudes perfectly ( eg h k 2l, and the rest approximately so the maps look reasonable. Have you run MR in all possible spacegroup for your point group? Eleanor On 13 October 2017 at 22:08, Gerard Bricogne <g...@globalphasing.com<mailto:g...@globalphasing.com>> wrote: Dear Randy, On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote: > Just to add to this point. The MR algorithms in Phaser are now able > to make better use of intensity data, which is particularly > important when you have any very weak data. Having weak data can’t > be avoided when you have serious anisotropy (or tNCS or a > combination of the two). Unfortunately, if you use amplitudes that > have been through the French & Wilson (truncate) algorithm, the real > variation in intensity is partially masked because the posterior > amplitude values are computed on the prior assumption that all the > reflections in a resolution shell have the same underlying intensity > distribution. Your "unfortunately" calsue may be the case with the UCLA server, but your statement is not true about what the STARANISO server (or STARANISO as invoked within autoPROC) does: as I already indicated in a reply to you a few months ago in this BB, the version of TRUNCATE in STARANISO applies the French & Wilson procedure with a prior Wilson probability whose expectation value for the intensity is modulated by the anisotropy of the dataset. This is clearly explained on the server - see http://staraniso.globalphasing.org/staraniso_about.html#step16 With best wishes, Gerard. -- > The UCLA server actually uses Phaser under the hood — what they add is to > turn the anisotropic B-values into suggested resolution limits in the > different directions. However, I don’t think they allow you yet to submit > intensities, which would be better. > > Best wishes, > > Randy Read > > ----- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +44 1223 > 336500<tel:%2B44%201223%20336500> > Wellcome Trust/MRC Building Fax: +44 1223 > 336827<tel:%2B44%201223%20336827> > Hills Road E-mail: > rj...@cam.ac.uk<mailto:rj...@cam.ac.uk> > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk<http://www-structmed.cimr.cam.ac.uk/> > > > On 13 Oct 2017, at 10:29, vincent Chaptal > > <vincent.chap...@ibcp.fr<mailto:vincent.chap...@ibcp.fr>> wrote: > > > > Dear Gia and Paul, > > > > about anisotropy, one point to keep in mind is that it is not necessarily > > linked to the difference in resolution limits. > > In fact I am at the moment working on one of these cases, with extremely > > large difference in resolution limits, but relatively low anisotropy. > > Anisotropy is more a deviation from "normal" intensity falloff as a > > function of resolution. There is a thin difference/relationship between the > > two concepts that I think is worth investigating. > > > > we have performed a statistical analysis of this phenomenon and the paper > > is in revision at the moment, but if you want to know where your anisotropy > > stands in respect to all the other PDBs out there, feel free to contact me > > off list. > > You mention MR: Phaser calculates the anisotropy so you can find the value > > in the first lines of the log. > > > > Staraniso or the UCLA server are good to test if you have anisotropy. > > Staraniso has a newer way of dealing with intensity falloff and accounting > > for it. > > > > All the best > > Vincent > > > > > > > > > > On 13/10/2017 10:58, Paul Miller wrote: > >> I had a similar problem to what you describe. In my case the dataset was > >> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were > >> stuck similar to yours but the map looked good. I was told by someone with > >> a much better appreciation of the theory than myself that the anisotropy > >> was causing the problem. > >> > >> It would be interesting to know from an expert in anisotropy e.g. the > >> creators of UCLA anisotropy server or Startaniso whether anisotropy can > >> cause this problem and whether there is any way around it. > >> > >> Cheers, Paul > >> > >> Paul Steven Miller (PhD) > >> Postdoctoral Researcher > >> University of Oxford > >> Wellcome Trust Centre for Human Genetics > >> Division of Structural Biology > >> Roosevelt Drive > >> Oxford > >> OX3 7BN > >> > >> > >> ---- Original message ---- > >> > >>> Date: Fri, 13 Oct 2017 10:30:22 +0200 > >>> From: CCP4 bulletin board > >>> <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf of > >>> Gianluca Cioci <gianluca.ci...@gmail.com<mailto:gianluca.ci...@gmail.com>> > >>> ) > >>> Subject: [ccp4bb] High R/Rfree after MR > >>> To: > >>> CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > >>> > >>> > >>> Dear All, > >>> I am trying to refine a structure at 3.3A. Model has > >>> 60% identity to the target. Maps look OK (for 3.3A) > >>> and rebuilding in Coot is relatively > >>> straightforward. However, after some rebuilding > >>> cycles the R factors are stuck at 0.37/0.39 > >>> (REFMAC). > >>> XTRIAGE tells me that everything is normal (no twin, > >>> 98% completeness, R=3.5% in the low resolution bin), > >>> perhaps some anisotropy is present. > >>> I have already refined 2 homologous structures at > >>> resolutions going from 3.2 to 3.8 and there were no > >>> problems (final R ~ 0.21/0.24). > >>> Any advice ? > >>> Thanks, > >>> GIA > >>> > > > > -- > > Vincent Chaptal, PhD > > Institut de Biologie et Chimie des Protéines > > Drug Resistance and Membrane Proteins Laboratory > > 7 passage du Vercors > > 69007 LYON > > FRANCE > > +33 4 37 65 29 01 > > http://www.ibcp.fr<http://www.ibcp.fr/> -- =============================================================== * * * Gerard Bricogne g...@globalphasing.com<mailto:g...@globalphasing.com> * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033<tel:%2B44-%280%291223-353033> * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889<tel:%2B44-%280%291223-366889> * * * =============================================================== ________________________________ UT Southwestern Medical Center The future of medicine, today.
