Dear Marcelo,

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.

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?

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.

Good luck!

Randy Read

On 7 Aug 2018, at 10:35, Marcelo Liberato <[log in to unmask]> wrote:

Dear CCP4 members, 

I need your help to figure out what is going on with my data. 
I've integrated my data set in space groups P1, P2 and P222. All with cell parameters: 45 89 149 90 90 90.

Aimless always indicates P212121 as the correct space group and the resolution is about 2.0 A.

I have tried MR (Phaser) with all these space groups (and the alternative ones) using a trimmed model (I've cut some loops) with 37 % identity (besides the low identity, this enzyme belongs to a protein family with high structural similarity). In most cases MR resulted in TFZ between 12-19 and LLG 200-400. 
However, refinement with refmac (with and without twin) generally results in Rwork/Rfree ~ 0.4400/0.4800. 
The best scenario was obtained when data was integrated in P21. MR resulted in TFZ=19 and LLG=450, and refmac (with twin) resulted in Rwork/Rfree = 0.4129/0.4517. This data was used in Autobuild (Phenix), twin law h,-k,-l,  resulting in Rwork/Rfree = 0.3525/0.4040. The final model seems to fit the electron density, but the map is not very good and there is a lot of bias. 

All data generated by aimless and MR in the different space groups were analyzed by Xtriage. In all cases, "Translational pseudo-symmetry is very likely present in these data" with an off-origin peak (38 % of origin peak) at fractional coordinates (0.0, 0.5, 0.13). 

Now, I am stuck and I have no idea how to solve this problem. Could you please help me with this?

The log files of aimless, MR, refmac and Xtriage of the "best scenario" are attached. 

Many thanks

Marcelo Liberato

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<xtriage_30.log><49_refmac5.log><46_phaser_MR.log><45_aimless.log>

------
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research      Tel: + 44 1223 336500
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