Thanks again Herman, The protein is a two domain protein (approx 40aa, 350aa split) - searching with either is proving fruitless.
Original, wild-type cell = 49 x 75 x 80 P212121 This painful mutant = 39 x 157 x 75 beta=98.26 P21 (so one can say that there seems to be a relationship there, wt b = mutant c, wt c = 2x mutant a?) We have been bitten in the past by crystallizing contaminants, but (touch wood) those are always small crystals one / drop, in a few conditions. This problem set has been replicated across at least 6 differing crystals (grown in different conditions), where there are many crystals / drop......along with the similar cell I'm confident that we are seeing diffraction from what we expect I will check the diffraction images more closely, but (does anyone agree here?) I find this sometimes obscured by modern fine-slice/Pilatus methodology Might be one for 2017! p.s. no anomalous signal in there - ironically mutant we're using knocks bound metal out! Thanks again Andy From: herman.schreu...@sanofi.com [mailto:herman.schreu...@sanofi.com] Sent: 19 December 2016 16:26 To: Andrew Lovering; CCP4BB@JISCMAIL.AC.UK Subject: AW: unusual monoclinic relation? Dear Andy, I don't think you will solve this pre-Xmas, but here are some more suggestions: -is there any relationship with the unit cell of the parent, unmutated protein? This might give some ideas of the packing of the problem crystals. -are some promising solutions being rejected due to clashes? In that case you might try to allow for more clashes. -can the protein be split in some separate domains? In that case you could try MR with the separate domains. -Are you sure the crystallized protein is the protein you think you crystallized? (see contamination database). -Check your diffraction images to make sure there are no pathologies present. Best, Herman Von: Andrew Lovering [mailto:a.lover...@bham.ac.uk] Gesendet: Montag, 19. Dezember 2016 12:30 An: Schreuder, Herman R&D/DE; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Betreff: RE: unusual monoclinic relation? Thanks Herman. In short: -no twinning suggested from xtriage etc -P2 doesn't give a solution either -monoclinic cell would have 2 (60% solvent) or 3 (40% solvent) copies -I originally thought the zanuda P1 route would be the way to go, but phaser was still churning away after running overnight and so I killed the job thinking it was thrashing Andy From: herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com> [mailto:herman.schreu...@sanofi.com] Sent: 19 December 2016 10:43 To: Andrew Lovering; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: AW: unusual monoclinic relation? Dear Andrew, Just a few questions: -Do the processing/refinement programs suggest twinning? -Are you sure your space group is P21 and not P2? Did you try MR in P2? -How many protein molecules do you expect in the asymmetric unit? P2(1) is a very low symmetry space group. In this case I would not try to be clever and just reprocess the data in P1 and run MR in P1. With Zanuda you can afterwards try to figure out what the real space group could be. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Andrew Lovering Gesendet: Montag, 19. Dezember 2016 09:43 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Betreff: [ccp4bb] unusual monoclinic relation? Dear All, I have just collected data on a mutant of a protein that should be facile to solve by molrep (one residue/320 changed, approx 2Ang resolution) but is proving problematic. Data merging stats look good. The spacegroup is monoclinic, P21, the cell: a=39.47 b=157.36 c=74.9 beta=98.26 I spotted the relevant monoclinic twin laws on ccp4 twinning page and all relate multiples of axes a and c with one another (na +nc etc) but in the above case it would appear b~ = 4a There are other datasets, all index in this way, some hint at issues by indexing with the alternate a=74 b=157 c=79 (where a and c "swap" with a doubled, and thus our b=4a turns into b=2c) I would appreciate any advice on how to progress! Be nice to solve it pre-xmas..... Best & thanks in advance, Andy
