Hi Eleanor, Yes, of course you are correct about the beta~90° requirement for possible twinning here-I was mistakenly thinking about pseudomerohedry in higher symmetry space groups. The L plot looks like well-behaved data, with a straight line that closely tracks the model untwinned one. Pointless, provided with data integrated in P1, choses P212121 with high confidence (0.95-0-.99), which is similar to the results of analysis using xtriage in PHENIX. Unsymmetrized cell angles are within 0.02-0.05° of 90°. Finally, the protein we crystallized is (to-be-tested wrong protein scenario aside) identical to the previously crystallized one, and our unit cell axes are the same within 5% or so. Best regards, Mark
Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 [email protected] On 9/3/15 2:45 PM, "Eleanor Dodson" <[email protected]> wrote: >Disorder almost always produces streaked spots, but I guess it isn't >compulsory! > >By the way, you can have twinning in Monoclinic if B ~ 90. without having >a = c. > > >. What does the L plot look like? Have you used pointless which gives the >CC for each symmetry operator separately - sometimes that shows say the >00 l axis is more 2-fold ish than the 0k0 axis.. > > > >Eleanor >PS - If the related protein fits into a similar cell with the same SG is >yours bigger? > > > > >On 3 September 2015 at 18:56, Mark Wilson ><[email protected]> wrote: > >Dear Remy, >Indeed, I think you may be correct and we're pursuing this now. A perfect >0.5 lattice translocation along b in P212121 would (I think) result in the >pathology we observe. We do not see zones of streaked reflections in the >images, but my thinking is that if the lattice defect is coincident with a >crystallographic translation operator, perhaps we wouldn't expect to. >Best regards, >Mark > >Mark A. Wilson >Associate Professor >Department of Biochemistry/Redox Biology Center >University of Nebraska >N118 Beadle Center >1901 Vine Street >Lincoln, NE 68588 >(402) 472-3626 <tel:%28402%29%20472-3626> >[email protected] > > > > > > >On 9/3/15 12:49 PM, "Remy Loris" <[email protected]> wrote: > >>Dear Mark, >> >>My suspicion is that what you observe here is a lattice disorder, >>possibly related to what is described in >> >>Jimin Wang, Satwik Kamtekar, Andrea J. Berman and Thomas A. Steitz >>(2005) Correction of X-ray intensities from single crystals containing >>lattice-translocation defects Acta Cryst D61, 67-74. >> >>If your unit cell is offset statistically by 0.5 in the b-direction, >>this should provide such a strong non-origin peak as you observe. In the >>cases that have been described until now, this type of disorder also >>involves zones of nice sharp reflections and other zones with more >>streaky reflections. Do you see something similar? >>In order to use such data, they have to be corrected as described in the >>paper above. >> >>Possibly, the crystals with the 10% non-origin peak also have the >>disorder, but much less pronounced so that omitting the required >>correction did not prevent structure determination and refinement >>(similar to let say a small fraction of merohedral twinning that is >>overlooked). >> >>Remy Loris >>Vrije Universiteit Brussel and VIB >> >>DOI: 10.1107/S0907444904026721 >> >>On 03/09/15 18:13, George Sheldrick wrote: >>> Dear Mark, >>> >>> Since your resolution is good enough, perhaps you should try to solve >>> it ab initio with Arcimboldo Lite. This has already solved a number of >>> structures that turned out to be unexpected. >>> >>> Best wishes, George >>> >>> >>> On 09/03/2015 05:44 PM, Mark Wilson wrote: >>>> Hi Herman, >>>> A fair point-the odds of "depressing coincidence" do seem to be >>>> climbing! >>>> We did inspect the deposited data for a similar peak and, while one is >>>> present, it is only ~10% of the origin and at a different location. >>>> We'll >>>> do some due diligence on our end by re-dissolving crystals and >>>> performing >>>> mass spec. As there seems to be some interest in this, I'll update >>>>once >>>> we've figured it out, even if it's an embarrassing case of wrong >>>> protein, >>>> same cell. >>>> Best regards, >>>> Mark >>>> >>>> Mark A. Wilson >>>> Associate Professor >>>> Department of Biochemistry/Redox Biology Center >>>> University of Nebraska >>>> N118 Beadle Center >>>> 1901 Vine Street >>>> Lincoln, NE 68588 >>>> (402) 472-3626 >>>> [email protected] >>>> >>>> >>>> >>>> >>>> >>>> >>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of >>>> [email protected]"<[email protected] on behalf of >>>> [email protected]> wrote: >>>> >>>>> Dear Mark, >>>>> >>>>> In this case you will have to apply Baysian statistics: given the >>>>> prior: >>>>> same protein, same space group same cell dimensions and molecular >>>>> replacement fails completely, the likelihood of having some >>>>>depressing >>>>> coincidence somewhere is approaches 100%! >>>>> >>>>> What I would do in addition to excellent suggestions you already >>>>> got, is >>>>> to try to download the Fobs from the pdb for the structures with the >>>>> same >>>>> protein, space group and cell dimensions, and calculate pattersons >>>>>with >>>>> those. Sometimes strong peaks appear in pattersons for no obvious >>>>> reasons. >>>>> I would also consider statistical disorder, which will not show up in >>>>> twinning statistics since in this case F's (including phases) are >>>>>added >>>>> instead of I's. Anyways, it will be an interesting puzzle to solve! >>>>> >>>>> Good luck, >>>>> Herman >>>>> >>>>> >>>>> -----Ursprüngliche Nachricht----- >>>>> Von: CCP4 bulletin board [mailto:[email protected]] Im Auftrag >>>>>von >>>>> Mark Wilson >>>>> Gesendet: Donnerstag, 3. September 2015 02:06 >>>>> An: [email protected] >>>>> Betreff: Re: [ccp4bb] Translational NCS with one molecule in ASU >>>>> >>>>> Dear CCP4 Community, >>>>> I've had a number of helpful responses (on- and off-list) that I will >>>>> briefly summarize via response, including information that I probably >>>>> should have included in the original post. Many have suggested a >>>>>wrong >>>>> space group, which I agree seems probable. MR was attempted in >>>>>PHASER >>>>> with all possible choices of space group for a primitive orthorhombic >>>>> lattice, and in all cases failed with no rotation or translation >>>>>peaks >>>>> above a Z-score of 5. >>>>> I've not yet tried monoclinic lattices and will, but this still >>>>> wouldn't >>>>> explain (to me anyway) an apparently impossible combination of >>>>> translational NCS in P212121 with a cell that can't accommodate a >>>>> second >>>>> molecule unless twinning was also present, which may be the case (as >>>>> Eleanor suggested). Others have asked about evidence of missed weak >>>>> reflections indicating a larger true cell, which I looked for but >>>>> didn't >>>>> see in these images. The crystal that was used was mounted at room >>>>> temperature, so there is no opportunity for cryo artifacts to have >>>>>done >>>>> something strange to the cell. >>>>> Other suggestions included the presence of strong internal >>>>> symmetry in >>>>> the molecule, which is present, but as a pseudo-threefold, which >>>>>seems >>>>> incompatible with my NCS centering operation. One respondent >>>>>suggested >>>>> that we've crystallized the wrong molecule, which is something I also >>>>> worried about a bit. Although possible, the space group and cell >>>>> for our >>>>> crystal are both as previously reported for this protein by another >>>>> group, so it would be a depressing coincidence if we crystallized the >>>>> wrong protein in the same cell. I'll be happy to update if/when we >>>>> figure >>>>> this out should it be of interest to the board. Thank you all for >>>>>your >>>>> thoughtful responses, which arrived in impressive number in the time >>>>>it >>>>> took me to drive home. >>>>> Best regards, >>>>> Mark >>>>> >>>>> Mark A. Wilson >>>>> Associate Professor >>>>> Department of Biochemistry/Redox Biology Center University of >>>>>Nebraska >>>>> N118 Beadle Center >>>>> 1901 Vine Street >>>>> Lincoln, NE 68588 >>>>> (402) 472-3626 <tel:%28402%29%20472-3626> >>>>> [email protected] >>>>> >>>>> >>>>> >>>>> >>>>> >>>>> On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor Dodson" >>>>> <[email protected] on behalf of >[email protected]> wrote: >>>>> >>>>>> Well - a translation of 0 0.5 0 would generate absences along b so >>>>>> that the SG could be P212121 or P 21 2 21Š >>>>>> >>>>>> >>>>>> I would suspect twinning and a monoclinic SG . >>>>>> Or as we found sadly - half the protein had disappeared in the >>>>>> crystallisation trials.. >>>>>> >>>>>> >>>>>> But such a translation must mean you almost have a halved unit cell? >>>>>> Another way of saying there isn't enough room for your molecule.. >>>>>> >>>>>> >>>>>> >>>>>> On 2 September 2015 at 22:38, Shane Caldwell >>>>>> <[email protected]> wrote: >>>>>> >>>>>> Are you certain it's actually P212121? One possibility is you're at >>>>>> lower symmetry and the Patterson peak corresponds to the NCS between >>>>>> particles that are almost-but-not-quite crystallographically >>>>>> equivalent. In that case, MR probably wouldn't work. Does >>>>>> searching in >>>>>> P1 find anything? >>>>>> >>>>>> Shane Caldwell >>>>>> >>>>>> McGill University >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> On Wed, Sep 2, 2015 at 5:19 PM, Mark Wilson<[email protected]> >>>>>>wrote: >>>>>> >>>>>> Dear CCP4 community, >>>>>> I've encountered a curious problem with some recently collected >>>>>>data. >>>>>> I have a 1.8 Å resolution dataset that scales well in P212121 (log >>>>>> file >>>>>> available upon request). The unit cell parameters are similar to >>>>>> those >>>>>> reported for crystals of the same protein in a previous publication, >>>>>> although my crystallization condition is different. Nevertheless, >>>>>>my >>>>>> data produce a strong (47% of origin) peak in the Patterson map at >>>>>> 0.0, >>>>>> 0.5, 0.0, indicative of translational NCS. However, the unit cell >>>>>> parameters can accommodate only one molecule in the ASU without >>>>>> single-digit solvent content. Moreover, molecular replacement with >>>>>>a >>>>>> model that should be nearly identical fails. Standard >>>>>>intensity-based >>>>>> tests show no evidence of twinning or other data pathology. Any >>>>>> thoughts would be appreciated. >>>>>> Best regards, >>>>>> Mark >>>>>> >>>>>> Mark A. Wilson >>>>>> Associate Professor >>>>>> Department of Biochemistry/Redox Biology Center University of >>>>>>Nebraska >>>>>> N118 Beadle Center >>>>>> 1901 Vine Street >>>>>> Lincoln, NE 68588 >>>>>> (402) 472-3626<tel:%28402%29%20472-3626> >[email protected] <mailto:[email protected]> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>> >>> >> > > > > > > > >
