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: [email protected] [mailto:[email protected]] > Sent: 19 December 2016 16:26 > To: Andrew Lovering; [email protected] > 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:[email protected]] > Gesendet: Montag, 19. Dezember 2016 12:30 > An: Schreuder, Herman R&D/DE; > [email protected]<mailto:[email protected]> 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: [email protected]<mailto:[email protected]> > [mailto:[email protected]] Sent: 19 December 2016 10:43 > To: Andrew Lovering; [email protected]<mailto:[email protected]> > 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:[email protected]] Im Auftrag von > Andrew Lovering Gesendet: Montag, 19. Dezember 2016 09:43 > An: [email protected]<mailto:[email protected]> > 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
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