Thanks Tim, Yes, I may give that a go next time (the wild-type was actually solved by S-SAD, very nice ordered bridge)
Or....perhaps time to seed / derivitize / mutate and get different packing. Still, not before xmas! Andy -----Original Message----- From: Tim Gruene [mailto:tim.gru...@psi.ch] Sent: 19 December 2016 19:58 To: Andrew Lovering Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] unusual monoclinic relation? Dear Andrew, did you remove all cysteins, and all methionines with the mutations? Your resolution is about 2A, if I understand correctly. This may be suitable for S- SAD. I would try to get access to a modern inhouse machine to get high quality data at high multiplicity. Some modern synchrotron beamlines offer more than 1-circle goniometers, but good quality data is more easily collected with a multi-circle instrument. Best, Tim On Monday, December 19, 2016 4:42:00 PM CET Andrew Lovering wrote: > 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 -- -- Paul Scherrer Institut Tim Gruene - persoenlich - OFLC/102 CH-5232 Villigen PSI phone: +41 (0)56 310 5297 GPG Key ID = A46BEE1A
