Hi Eleanor, I agree that experimental phases may be needed, although I may take the lazy way out and just aggressively screen for a less problematic crystal form. 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 mwilso...@unl.edu On 9/3/15 4:06 PM, "CCP4 bulletin board on behalf of Eleanor Dodson" <CCP4BB@JISCMAIL.AC.UK on behalf of eleanor.dod...@york.ac.uk> wrote: >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 ><adrian.gold...@helsinki.fi> 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 <mwilso...@unl.edu> 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 >> mwilso...@unl.edu >> >> >> >> >> >> >>> On 9/3/15 2:45 PM, "Eleanor Dodson" <eleanor.dod...@york.ac.uk> 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 >>> <mwilso...@unl.edu> 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> <tel:%28402%29%20472-3626> >>> mwilso...@unl.edu >>> >>> >>> >>> >>> >>> >>>> On 9/3/15 12:49 PM, "Remy Loris" <relo...@vub.ac.be> 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 >>>>>> mwilso...@unl.edu >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> On 9/3/15 10:25 AM, "CCP4 bulletin board on behalf of >>>>>> herman.schreu...@sanofi.com"<CCP4BB@JISCMAIL.AC.UK on behalf of >>>>>> herman.schreu...@sanofi.com> 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:CCP4BB@JISCMAIL.AC.UK] Im Auftrag >>>>>>> von >>>>>>> Mark Wilson >>>>>>> Gesendet: Donnerstag, 3. September 2015 02:06 >>>>>>> An: CCP4BB@JISCMAIL.AC.UK >>>>>>> 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> >>>>>>><tel:%28402%29%20472-3626> >>>>>>> mwilso...@unl.edu >>>>>>> >>>>>>> >>>>>>> >>>>>>> >>>>>>> >>>>>>> On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor >>>>>>>Dodson" >>>>>>> <CCP4BB@JISCMAIL.AC.UK on behalf of >>> eleanor.dod...@york.ac.uk> 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 >>>>>>>> <shane.caldwel...@gmail.com> 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<mwilso...@unl.edu> >>>>>>>> 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> >>> mwilso...@unl.edu <mailto:mwilso...@unl.edu> >> > > > > > > >
