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Dear Vaheh and Phil, Sorry, I was a bit misleading – antibody fragment I used is a nanobody (VHH, 15kDa). I was worried about the cell content because when I started the pdb deposition, there was a warning that solvent content is expected to be below 80%, so I thought maybe I missed a correct solution. But if that’s acceptable value the problem is solved. Thank you for the quick answer and the suggestion about the resolution! Best regards, Anna Dr. Anna Koromyslova, Postdoctoral researcher German Cancer Research Center (DKFZ), F150 Im Neuenheimer Feld 242 D-69120 Heidelberg Germany From: CCP4 bulletin board <[log in to unmask]> on behalf of "Oganesyan, Vaheh" <[log in to unmask]> Reply-To: "Oganesyan, Vaheh" <[log in to unmask]> Date: Tuesday, July 11, 2017 at 7:05 PM To: "[log in to unmask]" <[log in to unmask]> Subject: Re: [ccp4bb] Problem with a cell content 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:[log in to unmask]] On Behalf Of Koromyslova, Anna Sent: Tuesday, July 11, 2017 12:32 PM To: [log in to unmask] 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. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.