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How similar is this data set to the previously crystallised one? I think you said there is no translation vector in the previous one so they can't be very similar. Maybe it is time for experimental phases! Eleanor On 3 September 2015 at 21:47, Adrian Goldman <[log in to unmask]> wrote: > This would be my feeling too - one real 21, a twin axis and > pseudosymmetry. The standard perfect storm. > > Sent from my iPhone > > > On 3 Sep 2015, at 21:07, Mark Wilson <[log in to unmask]> wrote: > > > > 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 > > [log in to unmask] > > > > > > > > > > > > > >> On 9/3/15 2:45 PM, "Eleanor Dodson" <[log in to unmask]> 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 > >> <[log in to unmask]> 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> > >> [log in to unmask] > >> > >> > >> > >> > >> > >> > >>> On 9/3/15 12:49 PM, "Remy Loris" <[log in to unmask]> 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 > >>>>> [log in to unmask] > >>>>> > >>>>> > >>>>> > >>>>> > >>>>> > >>>>> > >>>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of > >>>>> [log in to unmask]"<[log in to unmask] on behalf of > >>>>> [log in to unmask]> 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:[log in to unmask]] Im Auftrag > >>>>>> von > >>>>>> Mark Wilson > >>>>>> Gesendet: Donnerstag, 3. September 2015 02:06 > >>>>>> An: [log in to unmask] > >>>>>> 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> > >>>>>> [log in to unmask] > >>>>>> > >>>>>> > >>>>>> > >>>>>> > >>>>>> > >>>>>> On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor Dodson" > >>>>>> <[log in to unmask] on behalf of > >> [log in to unmask]> 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 > >>>>>>> <[log in to unmask]> 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<[log in to unmask]> > >>>>>>> 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> > >> [log in to unmask] <mailto:[log in to unmask]> > > >