<html><head></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><div><div><div><font class="Apple-style-span" size="4"><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">I have a question of general significance: in what resolution NCS restrains and Rfree become IRRELEVANT?</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">Axel Brunger invented Rfree to save our necks from refining garbage into the structure distantly looking like protein.</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">Since than Rfree was idolized. However, there is a big difference between structures at 4.1 Angstrom and 1.4 Angstrom. </div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">In small molecule crystallography we can easily achieve 10 or 20 observations per refined parameter (depends on presence or absence of inversion center), therefore, no one care about Rfree in the small molecules community.</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">In the well ordered protein structures, the bulk water region is working against us lowering diffraction strength contributing to 1/Volume, but it is also on our side minimizing a volume occupied </div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">by protein molecules (less atoms, fewer parameters). I have a structure (not yet published) where for 18000 protein atoms and about 9000 other atoms (water molecules, sulfate ions, sugars from cryo-protection etc)</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">there are 750,000 independent observations. It makes about 28 observations per atom and together with the chemical observations such as bonds and angles which rarely differs from their classical values defined by small structures, if we keep anomalous data properly scaled and separated (there will be differences in good data sets that depends on S atoms and some other ions in solute, or even oxygen atoms) -</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">we have quite good ratio of observations per refined parameter.</div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; font: normal normal normal 14px/normal Helvetica; ">So my question is: Do WE and WHAT FOR need to mess with Rfree in structures of relatively/very high resolutions? </div><div><br></div></font></div><div apple-content-edited="true">
Dr Felix Frolow <br>Professor of Structural Biology and Biotechnology<br>Department of Molecular Microbiology<br>and Biotechnology<br>Tel Aviv University 69978, Israel<br><br>Acta Crystallographica F, co-editor<br><br>e-mail: <a href="mailto:mbfrolow@post.tau.ac.il">mbfrolow@post.tau.ac.il</a><br>Tel: ++972-3640-8723<br>Fax: ++972-3640-9407<br>Cellular: 0547 459 608
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<br><div><div>On Jan 31, 2012, at 17:35 , Pavel Afonine wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"><div>Hi Simon,<br><br>the difference is well illustrated in<br><br>F. Fabiola, A. Korostelev and M. S. Chapman<br>Acta Cryst. (2006). D62, 227-238<br>Bias in cross-validated free R factors: mitigation of the effects of non-crystallographic symmetry<br><br>The question is whether we can reproduce it in the exact same set of test structures.<br><br>Pavel<br><br>On 1/31/12 1:46 AM, Simon Kolstoe wrote:<br><blockquote type="cite">Thanks for the interesting comments.<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">I was just wondering what sort of "difference" we are expecting to see? Is it just a case of preventing an artificially lowered Rfree or is there an expectation to see a difference in the quality of the electron density?<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">Simon<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">---------------------------------------------------------------<br></blockquote><blockquote type="cite">Dr Simon Kolstoe<br></blockquote><blockquote type="cite">Laboratory for Protein Crystallography<br></blockquote><blockquote type="cite">Wolfson Drug Discovery Unit<br></blockquote><blockquote type="cite">University College London<br></blockquote><blockquote type="cite">Rowland Hill Street, London NW3 2PF<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">Tel: 020 7433 2765<br></blockquote><blockquote type="cite"><a href="http://www.ucl.ac.uk/~rmhasek">http://www.ucl.ac.uk/~rmhasek</a><br></blockquote><blockquote type="cite">---------------------------------------------------------------<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">On 31 Jan 2012, at 08:47, A Leslie wrote:<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite"><blockquote type="cite">Hi Randy,<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"> I can't remember if I ever mentioned this to you, but when I was working on the HepB capsid structure (30 fold ncs if i remember correctly) I tried using a "thin shell within a thick shell" method of selecting Rfree, to avoid the issue that within a thin shell there are still relationships between those reflections within the shell and those just outside it. I forget the details, but I think I used a thin shell of 1-2 rlps wide for the reflections to be used for Rfree, but I also excluded from the refinement reflections within a thick shell 4-5 rlps wide (the thin shell was in the middle of the thick shell). Because this excluded so many reflections I could only have 3 thick/thin shells altogether, so I chose them at low, middle and highish resolution.<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">The upshot of all this was that it was no help at all. Almost regardless of, say, the relative weight I put on the Xray terms, or anything else I did, I could never get the Rfree to go up ! The strict NCS restraints were so strong that the refinement essentially always "behaved".<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">This for me destroyed all my faith in this thin shell idea !<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">So this is definitely NOT an example where it worked.<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">I have not sent this to the bulletin board because my memory of exactly what I did is a bit hazy, but the message was clear enough.<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">Cheers<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">Andrew<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">On 30 Jan 2012, at 17:06, Randy Read wrote:<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">I'd be meaning to contribute to this debate, and now that I see my name mentioned...<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">I used to be a very strong believer in selecting the cross-validation data in thin shells, when you have NCS. I even had a recollection (a case of false memory syndrome, it seems) that we did this for our own case of 20-fold NCS, i.e. four copies of the Shiga-like toxin B-subunit pentamer cocrystallized with the Gb3 trisaccharide (Ling et al, 1998).<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">As a believer in thin shells, I was trying to convince Pavel to put an option for this in Phenix (like the one in sftools). He said that he'd never seen any evidence that it was necessary or made any difference. So I went back to the Shiga-like toxin structure and started parallel refinements from the MR solution, either choosing the cross-validation data randomly or in thin shells. And, guess what, I couldn't see any significant difference in how well the refinement went, even though I was pretty certain before doing that experiment that it would make a big difference. In fact, both refinements went pretty well.<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">So if thin shells aren't necessary even in an extreme case of NCS, then I suspect that they're not that useful in the more usual case of lower-order NCS.<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">In any case, there is a problem even with the thin shells (which Bart Hazes pointed out even as he implemented it in sftools). The theory suggests that reflections within some distance in reciprocal space of some reflection or a point related to it by an NCS rotation should be correlated to the original reflection. All the points related by rotation will fall into the same resolution shell but, since the reciprocal-space distance is related to the inverse of the diameter of the molecule, the shell would have to have some thickness, and the reflections at the edge of the shell would still be correlated to reflections not in the shell. So even thin-shell cross-validation doesn't get around all the theoretical problems.<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">I'd be interested if someone has an example where it really does make a difference, but in the meantime it's hard to argue with Pavel's point of view!<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Regards,<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Randy<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">On 30 Jan 2012, at 15:26, Nathaniel Echols wrote:<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">On Mon, Jan 30, 2012 at 3:43 AM, Simon Kolstoe<<a href="mailto:s.kolstoe@ucl.ac.uk">s.kolstoe@ucl.ac.uk</a>> wrote:<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">I see from a quick google that it is possible to pick my Rfree's using thin resolution shells (coz I've got 20 fold NCS), however as I am someone who tries to avoid the GUI where at all possible,<br></blockquote></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Why? Some things are simply easier to do in the GUI, or at least more<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">obvious - otherwise we wouldn't bother writing one.<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">could someone let me know what the command line way of doing this is?<br></blockquote></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">In phenix.refine, you probably want something like this (some<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">parameters optional, but the defaults are probably not what most<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">people expect):<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">xray_data.r_free_flags.generate=True<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">xray_data.r_free_flags.fraction=0.05<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">xray_data.r_free_flags.max_free=None<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">xray_data.r_free_flags.use_dataman_shells=True<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">xray_data.r_free_flags.n_shells=20<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Randy and Paul claim that this doesn't help very much with the NCS<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">issue, however.<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">-Nat<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">_______________________________________________<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">phenixbb mailing list<br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><a href="http://phenix-online.org/mailman/listinfo/phenixbb">http://phenix-online.org/mailman/listinfo/phenixbb</a><br></blockquote></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">------<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Randy J. Read<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Department of Haematology, University of Cambridge<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Cambridge Institute for Medical Research Tel: + 44 1223 336500<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Wellcome Trust/MRC Building Fax: + 44 1223 336827<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Hills Road E-mail: <a href="mailto:rjr27@cam.ac.uk">rjr27@cam.ac.uk</a><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">_______________________________________________<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite">phenixbb mailing list<br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><blockquote type="cite"><a href="http://phenix-online.org/mailman/listinfo/phenixbb">http://phenix-online.org/mailman/listinfo/phenixbb</a><br></blockquote></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">_______________________________________________<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite">phenixbb mailing list<br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><a href="http://phenix-online.org/mailman/listinfo/phenixbb">http://phenix-online.org/mailman/listinfo/phenixbb</a><br></blockquote></blockquote><blockquote type="cite"><blockquote type="cite"><br></blockquote></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">_______________________________________________<br></blockquote><blockquote type="cite">phenixbb mailing list<br></blockquote><blockquote type="cite"><a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br></blockquote><blockquote type="cite"><a href="http://phenix-online.org/mailman/listinfo/phenixbb">http://phenix-online.org/mailman/listinfo/phenixbb</a><br></blockquote><br>_______________________________________________<br>phenixbb mailing list<br><a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br>http://phenix-online.org/mailman/listinfo/phenixbb<br></div></blockquote></div><br></div></div></body></html>