Dear Chris, I have looked at the images. The 1.65Å data set seems to have a high mosaicity, but to be otherwise ok. The 1.9Å seems to have one extremely long axis almost perpendicular to the rotation axis. One should always try to have such an axis parallel to the rotation axis during data collection. Here are some comments:
-For high mosaicity crystals, I would definitively try to process with XDS. In this case I find XDS much better than the other programs. -In cases like this I sometimes use a somewhat dirty trick in that I specify a much lower mosaicity (say half the refined value) and do not allow the processing software to refine the mosaicity. In this way, only the strong centers of the reflections are integrated and not the weaker flanks where most of the overlaps occur. An additional advantage is that less reflections get rejected. In some cases, this worked very well for me. -Did you process the 1.9Å data with one very long axis? Occaisonally, autoindexing may come up with axes with are only half the true axes. - Some of the "well-resolved" reflections in your "good" 1.9Å image may in fact be a pile-up of several reflections since in this orientation the very long axis may be parallel to the X-ray beam. Here I would carefully inspect the integration boxes and if necessary, make them as narrow as possible in the direction of the long axis. -It may be a good idea to start autoindexing with some of the "bad" images, since here the long axis is clearly visible. I am pretty sure that the poor quality of your 1.9Å maps is due to problems with the data processing. To run XDS,it may be easeier to run it via the CPP4 procedure XIA2, or via autoPROC from Global Phasing. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Chris Fage Gesendet: Sonntag, 23. Februar 2014 23:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] High Rwork/Rfree vs. Resolution Thanks again for the advice, everyone. As suggested, I tried NCS and TLS in phenix.refine, although my R-factors did not budge. I am now giving PDB_REDO and simulating annealing in PHENIX a shot. I am also looking into setting up XDS. Forgive my ignorance, but I am not sure how to check whether the bulk solvent model is reasonable. For these crystals, HKL2000 does invariably report high mosaicity along one axis (it is in the "red"). Yes, the structure was solved by MR. For the 1.65-angstrom map, the model is very complete, with density missing only for the N-terminal 6xHis tag and first three residues, as well as 5-10 other residues on flexible loops (the protein is ~300 residues, including the tag). Most side chains are well resolved. The quality of the 1.90-angstrom map is lower, with more gaps, more noise, and less side-chain coverage. In each map, there is no remaining density that legitimately needs to be filled. I have attached representative frames and relevant details from the HKL2000 scale logs. (Note that the 1.65-A set was originally scaled to 1.53 A.) As for making the datasets available before publication, I would have to check with my supervisor. The idea might not fly with him, as the structure is expected to be of relatively high impact. Best, Chris On Sat, Feb 22, 2014 at 3:00 AM, Francis Reyes <francis.re...@colorado.edu<mailto:francis.re...@colorado.edu>> wrote: > > 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
