Hi,

Looking at the pointless logfile for P222, there’s excellent evidence for a 2(1) screw along the shortest and longest cell edges as Eleanor says, which would make the space group P 21 2 21 for the data set used in the Phaser run with the data merged in P222.  The merging statistics are equally good for all 3 2-folds and for the identity, which implies, as Eleanor says, that the data most likely should be merged as orthorhombic.  If the data were twinned that would explain merging in higher symmetry even if the true space group were some version of P2 or P21, but the twinning statistics look pretty close to what one would expect for an untwinned crystal.

Maybe you’ve run Phaser in all 8 possible orthorhombic space groups in some other job, but in the job that the file name implies was run in P212121, it was actually only run in P222, i.e. the point group in which the data were merged.  So this job has missed all the more likely space groups.

What Phaser flags as an outlier rejection changed a couple of years ago, though we still haven’t published this.  For a long time, Phaser has rejected improbably large structure factors, i.e. ones that would be expected to occur less than one time in a million according to the Wilson distribution.  For about 2 years now, it has also been ignoring reflections flagged as containing very little information about the true intensity, i.e. ones in which the standard deviation of the intensity (SIGI) is large compared to the Wilson expected intensity.  When a crystal has very high anisotropy (as in this case, where the anisotropic delta B, or the difference between the weakest and strongest directions, is nearly 60 A^2, many of the reflections in the weak directions in reciprocal space will contain so little information that they can be ignored in the calculation (because they would contribute almost nothing apart from raising the CPU time!).  The presence of tNCS introduces more reflections that are systematically very weak, as well.  I should probably change the log file output to separate the counts for the rejections vs the ones being ignored.  There are data sets where nearly half of the reflections end up being ignored, so I wouldn’t worry about that for this data set.

Best wishes,

Randy Read

-----
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research    Tel: +44 1223 336500
Wellcome Trust/MRC Building                         Fax: +44 1223 336827
Hills Road                                                            E-mail: [log in to unmask]
Cambridge CB2 0XY, U.K.                               www-structmed.cimr.cam.ac.uk

> On 11 Aug 2018, at 20:36, Eleanor Dodson <[log in to unmask]> wrote:
> 
> Marcelo - there is something very wrong with the data. You dont to reprocess in other space groups - the P2/mmm symmetry looks convincing but PHASER says there are 3500 rejections! That is an awful lot - 10 is a more normal value. 
> I cant see the image clearly but something is causing problems. Split crystal? How are you processing it?
> 
> The Moments go crazy at high resolution. Maybe try redoing the MR & refinement  all at 3A? 
>  
> 
> The MR rotation function solution is wonderful, but you need to conside spacegroups P 21 2 21. and P 21 21 21 .
> The translation vector of 0 0.5 0.13 would produce absences along the b axis. 
> The screw axes along a and c look safe. 
> 
> 
> 
> On 11 August 2018 at 19:36, Marcelo Liberato <[log in to unmask]> wrote:
> I am sorry. I forgot to attach the image.
> 
> Cheers
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato <[log in to unmask]> escreveu:
> Dear Eleanor,
> 
> Thanks for you answer. 
> Indeed, there are clear ice rings in the images (example attached). So, I integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for P2 (in two different cells) and P222 data. 
> I agree that MR seems very good (in all cases), but the final density maps are always bad. Maybe the data has problems that I am not dealing with. 
> 
> Kind regards
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson <[log in to unmask]> escreveu:
> This MR looks good to me, but there are serious flaws with the data. Your secon moment plot from the aimless log has most spectacular spikes which are always a BAD THING, and the Wilson plot is not very smooth either.. 
> 
> As Randy says, try to sort those problems out first.
> 
> Then you have this message:
> 
> 
> TRANSLATIONAL NCS:
> 
> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> A translation of 0.5 along B will generate pseudo-absences along b so you can be sure whether there is a scre axis or not..
> 
> The space group is most likely orthorhombic - these indicators are pretty convincing for P2/mmm - so I dont know why you have chosen P21 as the spacegroup? 
> 
> 
> Scores for each symmetry element
> 
> Nelmt  Lklhd  Z-cc    CC        N  Rmeas    Symmetry & operator (in Lattice Cell)
> 
>   1   0.917   8.18   0.82   61009  0.298     identity
>   2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l}, along original k
>   3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l}, along original l
>   4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l}, along original h
> 
> So my suggestions:
> Sort out data problems
> 
> Merge as P2/mmm 
> 
> Let MR search select the most likely spacegroup of the 8 possible.
> 
> You cant even limit the b axis to be a screw axis .
> 
> Your refinement behavior looks OK, but the maps will look bad with spurious reflections in the list..
> 
> Eleanor
> 
> 
> 
> 
> 
> On 10 August 2018 at 19:02, Eleanor Dodson <[log in to unmask]> wrote:
> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR solution! 
> 
> 
> 
> On 10 August 2018 at 18:42, Eleanor Dodson <[log in to unmask]> wrote:
> Can you attach the refinement log?  
> 
> Eleanor
> 
> On 10 August 2018 at 16:57, Marcelo Liberato <[log in to unmask]> wrote:
> Dear Randy, 
> 
> Thank you very much for answering. I followed your suggestions but, unfortunately, I couldn't get a reasonable electron density map after MR and refinement.
> 
> 
> First I would look at the data to see if you have ice rings, because the peak in mean intensity and second moment of the intensity at about 2.25A resolution suggests an ice ring problem.  If so, you should make sure you don't contaminate the data with spurious large intensities.
> 
> Indeed, the data has ice rings. At first, I required imosflm to remove ice rings, but it didn't happened. So, I re-processed the data in different space groups removing the ice rings.  
> 
> Second, the statistics (e.g. the second moments plot after tNCS correction in Phaser) would be consistent with a scenario in which you have pseudosymmetry along with a twin operator that parallels the pseudosymmetry.  If that's true, it's hard to be sure of the symmetry.  For instance, if the structure really is monoclinic, can you be sure you chose the correct axis to be the 2-fold?
>  
> I am not sure. However, I tried two possible axis to be the 2-fold and none of them gave me reasonable maps after MR and refinement.
>  
> 
> Since you have a good model that gives clear MR solutions even in P21, you can probably process the data in P1 and solve it with 8 copies in the unit cell.  Then you can look at the symmetry of the MR solution (e.g. in Zanuda) and see whether it obeys any higher symmetry than P1.
> 
> I processed data in P1. After MR (with 8 copies in the ASU), it resulted in TFZ=11.6 and LLG=1434. But the map is still bad and high Rwork and Rfree.
> According to Zanuda, the data should be P21:
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>    | >>   4   | P 1 21 1   | 68.6868  |  0.6289  |  0.5487  |  0.5523  |
>    ---------------------------------------------------------------------
>    |      1   | P 1        | 69.4151  |  0.6171  |  0.5471  |  0.5559  |
>    |      4   | P 1 21 1   | 69.3810  |    --    |  0.5482  |  0.5442  |
>    |     11   | P 21 21 21 | 52.0271  |    --    |  0.6107  |  0.6178  |
>    ---------------------------------------------------------------------
>    | <<   4   | P 1 21 1   | 69.3810  |    --    |  0.5482  |  0.5442  |
>    ---------------------------------------------------------------------
> I processed in P21 using two different unit cells, and MR resulted in TFZ=20.8 and LLG=511, and TFZ=56 and LLG=2867. However, again, no good maps and statistics.   
> 
> Best regards
> 
> Marcelo Liberato 
> 
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