Still seems to me that the resolution could and should be pushed a little further at least—CC1/2 is still high, completeness is good, I/sigma also is good. Why not extend a little further, say to where one of these values gets too low? Might improve the maps a bit.
JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Koromyslova, Anna Sent: Tuesday, July 11, 2017 3:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problem with a cell content Sorry for the confusion, NCS was found only in a dataset where the cell dimensions were twice bigger regardless of the sp, and with a dimer as a solution. The solvent content was the same. Peak Distance Vector 73.3% 143.8Å: FRAC +0.000 +0.000 -0.500 (ORTH -0.0 0.0 -143.8). There was no NCS in a dataset with a smaller unit cell and sp P6222. Thank you! Best regards, Anna Dr. Anna Koromyslova, Postdoctoral researcher German Cancer Research Center (DKFZ), F150 Im Neuenheimer Feld 242 D-69120 Heidelberg Germany From: Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> Date: Tuesday, July 11, 2017 at 8:17 PM To: "Koromyslova, Anna" <a.koromysl...@dkfz-heidelberg.de<mailto:a.koromysl...@dkfz-heidelberg.de>> Cc: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>> Subject: Re: [ccp4bb] Problem with a cell content So SG could be P31 2 2 What is the height of NCS vector v origin? Eleanor Look at hklview to see hk i sections. Obviously all l = odd will be weak. On 11 July 2017 at 19:12, Koromyslova, Anna <a.koromysl...@dkfz-heidelberg.de<mailto:a.koromysl...@dkfz-heidelberg.de>> wrote: Dear Eleanor, NCS translation vector = 0 0 -0.5 in the final sp P6222, and in P622 or C121. All had the same solution. The protein tends to form a homodimer and if I use P1 space group molecular replacement can find several dimers, there is no translational ncs found during MR, but still solvent cell content was similarly high. Within a protein monomer there are no similar domains. Best regards, Anna From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> Reply-To: Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> Date: Tuesday, July 11, 2017 at 7:29 PM To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] Problem with a cell content This is very strange . If you have a large non- crystallographic translation vector you would expect either to have two molecules in the asymmetric unit or your one molecule must have two very similar domains? What is the n-c translation vector? Could you have assigned too high symmetry ? SG maybe P 62? That could explain the packing clashes Z scores of 27 are pretty good. Eleanor On 11 July 2017 at 18:05, Oganesyan, Vaheh <oganesy...@medimmune.com<mailto:oganesy...@medimmune.com>> wrote: Похоже, что вы уже решили структуру. Почему вас беспокоит плотность ваших кристаллов? Кристаллы бывают разные. First of all Fab by itself is already almost 50 kDa, so complex with antigen should be more than 50 kDa. Because you already solved the structure calculate the molecular mass based on your pdb file and rerun Matthews with correct mass. New numbers may be quite a bit different. Good indication of relatively low crystal density and consequently loose packing is the resolution of your data set. If you did not throw away data beyond 2.9A I’d suggest use them all. The reflections are too valuable to throw away. If data beyond some resolution is weak then they will have low contribution to the structure. Best if you calculate electron density maps at different resolutions at the end of refinement, compare them and use resolution that makes difference. Regards, Vaheh 8-5851 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Koromyslova, Anna Sent: Tuesday, July 11, 2017 12:32 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Problem with a cell content Dear CCP4 members, I am working on a structure of a protein in complex with an antibody fragment (approx. 50kDa together). Molecular replacement with closely related proteins always comes up with one complex in the asymmetric unit, although MW of protein to which Matthews applies is 125kDa and corresponds to two complexes. Phaser gives two warnings: Large non-origin Patterson peak indicates that translational NCS is present. Solutions with Z-scores greater than 27.2 (the threshold indicating a definite solution) were rejected for failing packing test I couldn’t get a solution with two subunits although I have tried multiple combinations including only conserved parts of both proteins and different space groups including P1. Phenix Autobuild also yielded only one complex. So, the question is whether I can use that structure as is despite very high solvent content (80%) or should I try smth else. I would be very grateful for any suggestions. When the solution with a single complex is refined the statistics are the following: R-work 0.2129 R-free 0.2459 Matthews Coefficient: 6.22 Percentage Solvent: 80.22 Resolution range (Å) 48.34 - 2.9 (2.98 - 2.9) Space group P 62 2 2 Unit cell 167.45 167.45 143.538 90 90 120 Multiplicity 19.1 (18.3) Completeness (%) 99.44 (94.39) Mean I/sigma(I) 24.59 (2.71) Wilson B-factor 64.28 R-merge 0.1256 (1.186) R-meas 0.1291 CC1/2 0.999 (0.85) CC* 1 (0.959) Thank you very much for your help, Anna Dr. Anna Koromyslova, Postdoctoral researcher German Cancer Research Center (DKFZ), F150 Im Neuenheimer Feld 242 D-69120 Heidelberg Germany To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. 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