[ccp4bb] A crystal symmetry broken beyond repair?
I have three brief questions (number two should be the easy one).
1. Where could an extra electron density of an (physically impossible)
additional molecule in an otherwise perfect map come from? (even in a wrong
solution in a {probably} too high space group?)
2. I would like to see how my data looks like in a RvR plot. How can I make
one? (Acta Cryst.D62, 83-95.)
3. Is it possible to have an order/disorder crystal defect with a pure
rotational disorder, e.g. the unit cell translations are not affected by
the disorder, only the orientations of the molecules in separate layers?
Stefan
[ccp4bb] Research Technician
The Hoelz Laboratory at the California Institute of Technology has an open research technician position for an undergraduate student that will graduate this June with a B.Sc. degree in Biochemistry, Biophysics, Chemistry, or Biology. For details, see https://jobs.caltech.edu/postings/1513
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. This is bad. Large swaths of red circles during integration is bad. I believe (check the Denzo manual) this means overlaps and overlaps get thrown out. Thus you are getting lower completeness. Was your oscillation range too large? Crystal very mosaic? However this could be because of a poor crystal orientation matrix by HKL2000 which in some cases can be alleviated by mosflm and xds. (HKL2000 is much more manual, there's a lot of buttons, which means you can shoot yourself in the foot if you are not careful). I would be particularly interested in a resolution bin breakdown in the integration and merging statistics. (I/sig and rmerge). You might as well post the refinement statistics (r and rfree) by resolution bin as well. You have a smallish unit cell that shoots to high resolution and getting a reasonable completion of the low resolution bins is paramount. Post the completeness of the 20-10A bin. Is this molecular replacement? How complete is the model? Aside from the completeness of the model, how far is it from the target? You mentioned that some regions of your crystal had smeary spots. This is also bad, particularly if the errors are not random (I.e anisotropic along one axis). This will confuse ML refinement. Let's see a single frame of your data. Cheers, F
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
As the excellent tips that you got indicate, lower R-factors can be obtained by getting better data (better crystals, better data collection, better data processing) or better fitting, i.e. refinement. In this respect, I am impressed by the automatic data processing protocols now being implemented. Also, the automatic local NCS refinement in REFMAC seems very good for our recent structures. But I would really want to make a general comment - not ALL structures can be better than the average! There will always be structures with 5% higher R/Rfree than the average in the same resolution range. Sometimes this will be due to suboptimal refinement, but sometimes it may simply not be possible to get better crystals and better data. Better not necessarily in term of resolution, but in terms of disorders like you describe for your plate-shaped crystals. What I mean is that one should make all efforts to get better crystals and data and refine structures as well as possible, but sometimes it may not be possible to beat the average of the pdb and one should not get too hung up by that. These structures should also be deposited and published. On the other hand, these rules that R-factor should be a certain value at a certain resolution, may lead to suboptimal refinement. For example the thought my R-factor is already better than the average could be counterproductive and lead people to stop refinement prematurely. Sometimes a structure will have Rs better than the average for the resolution, but still better refinement could lower it further and this should then be done. I can think of an MR solution using a very homologous model that was refined at higher resolution, structures with high NCS, or simply certain rock-solid proteins... Another popular one is (was?) that Rfree should always be below 30%, while several important structures justifiably have Rfrees quite a bit higher (others perhaps have not been refined enough). So while comparing R/Rfree to the average of existing structures is useful, it may not necessarily be a sign that a structure is bad if your Rs are 5 % higher, not should your Rs being at or below the average be an excuse for stopping refinement too early. Fear that ones Rs are not low enough may even lead to certain forms of cheating, for example not keeping the Rfree reflections truly free. On 22 Feb 2014, at 01:41, Chris Fage wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
I agree with the general points that Mark makes. For the basic problem to find out whether the model quality (and R-values) is limited by the data quality, you can compare CCwork with CC* - if CCwork is significantly lower than CC*, then this means that either your model could in principle be improved (you have not yet found the right parameterization), or the data suffers from correlated systematic errors. So it would be good to try the XDS data processing. best, Kay On Sat, 22 Feb 2014 23:15:33 +0100, Mark van Raaij mjvanra...@cnb.csic.es wrote: As the excellent tips that you got indicate, lower R-factors can be obtained by getting better data (better crystals, better data collection, better data processing) or better fitting, i.e. refinement. In this respect, I am impressed by the automatic data processing protocols now being implemented. Also, the automatic local NCS refinement in REFMAC seems very good for our recent structures. But I would really want to make a general comment - not ALL structures can be better than the average! There will always be structures with 5% higher R/Rfree than the average in the same resolution range. Sometimes this will be due to suboptimal refinement, but sometimes it may simply not be possible to get better crystals and better data. Better not necessarily in term of resolution, but in terms of disorders like you describe for your plate-shaped crystals. What I mean is that one should make all efforts to get better crystals and data and refine structures as well as possible, but sometimes it may not be possible to beat the average of the pdb and one should not get too hung up by that. These structures should also be deposited and published. On the other hand, these rules that R-factor should be a certain value at a certain resolution, may lead to suboptimal refinement. For example the thought my R-factor is already better than the average could be counterproductive and lead people to stop refinement prematurely. Sometimes a structure will have Rs better than the average for the resolution, but still better refinement could lower it further and this should then be done. I can think of an MR solution using a very homologous model that was refined at higher resolution, structures with high NCS, or simply certain rock-solid proteins... Another popular one is (was?) that Rfree should always be below 30%, while several important structures justifiably have Rfrees quite a bit higher (others perhaps have not been refined enough). So while comparing R/Rfree to the average of existing structures is useful, it may not necessarily be a sign that a structure is bad if your Rs are 5 % higher, not should your Rs being at or below the average be an excuse for stopping refinement too early. Fear that ones Rs are not low enough may even lead to certain forms of cheating, for example not keeping the Rfree reflections truly free. On 22 Feb 2014, at 01:41, Chris Fage wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Projects and problems like this are clearly a justification for asking to deposit not only the results from data processing, but also the raw data frames. These would allow developers to improve the models underlying their algorithms, and to find those corner cases where the algorithms break. That would help everyone. Maybe you could make this dataset (and sequence) available for the forthcoming IUCr conference, as an example for a difficult dataset? (send email to Ed Collins or me) You would profit from the fact that experienced crystallographers do their best to make the most of your data. best, Kay On Fri, 21 Feb 2014 20:13:33 -0600, Chris Fage cdf...@gmail.com wrote: Thanks for the assistance, everyone. For those who suggested XDS: I forgot to mention that I have tried Mosfim, which is also better than spot fitting than HKL2000. How does XDS compare to Mosflm in this regard? I am not refining the high R-factor structure with NCS options. Also, my unit cell dimensions are 41.74 A, 69.27 A, and 83.56 A, so there isn't one particularly long axis. I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. Thanks, Chris On Fri, Feb 21, 2014 at 6:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
