[phenixbb] Twinning Refinement
Joseph Brock
joeylives2ride at hotmail.com
Thu Mar 25 22:13:07 PDT 2010
Dear Phenix Users,
I'm currently using version:
Version: 1.4
Release tag: 3
cctbx tag: 2008_12_07_1353
Platform: mac-intel-osx osx-10.5.6
I am working on characterising substrate/inhibitor interactions of a protein, the structure has been solved many times so its unit cell dimensions, contents (1 molecule per ASU) and space group (P3121) are well characterized.
However, I have recently encountered several crystals that appear exhibit merohedral twinning, and am having trouble converging the R-factors to reasonable levels. The details of one such data set is described below:
Diffraction to ~1.9A was recorded using a high and low resolution pass of 180º each. When processed with HKL2000 separately or together, Scalepack wanted to reject huge amounts of my data (~20%). I was forced to increase the error scale factor to 2 and adjust my error model to the very high R-factors of the low-resolution shells. The summary table from my final round of scalepack is:
Shell Lower Upper Average Average Norm. Linear Square
limit Angstrom I error stat. Chi**2 R-fac R-fac
50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424
4.09 3.25 3885.1 568.9 64.1 1.410 0.416 0.480
3.25 2.84 1609.7 225.0 26.1 1.227 0.451 0.517
2.84 2.58 807.9 97.1 25.3 1.958 0.439 0.561
2.58 2.39 496.6 59.7 22.0 1.060 0.257 0.268
2.39 2.25 335.5 53.0 18.1 0.717 0.275 0.238
2.25 2.14 221.7 19.0 17.4 2.389 0.354 0.298
2.14 2.05 143.1 19.5 17.5 1.537 0.459 0.351
2.05 1.97 87.0 20.2 17.1 1.079 0.663 0.480
1.97 1.90 54.6 20.3 16.7 1.073 0.990 0.696
All reflections 1588.3 269.2 41.9 1.350 0.400 0.444
In addition, many systematic absences appear to be present:
Intensities of systematic absences
h k l Intensity Sigma I/Sigma
0 0 5 177.7 56.7 3.1
0 0 7 255.5 70.6 3.6
0 0 8 264.8 73.2 3.6
0 0 10 595.0 164.2 3.6
0 0 11 2343.5 1289.9 1.8
0 0 13 321.3 125.4 2.6
0 0 14 66.5 21.8 3.1
0 0 16 238.0 76.3 3.1
0 0 17 394.3 109.1 3.6
0 0 19 13.0 7.1 1.8
0 0 20 650.1 179.5 3.6
0 0 22 199.6 55.6 3.6
0 0 23 953.9 372.0 2.6
0 0 25 192.2 62.2 3.1
0 0 26 1038.7 405.0 2.6
0 0 28 96.3 28.9 3.3
0 0 29 2303.0 897.0 2.6
0 0 31 12.0 11.5 1.0
0 0 32 1622.4 516.5 3.1
0 0 34 11.5 13.8 0.8
0 0 35 22.8 14.8 1.5
0 0 37 27.9 18.0 1.5
0 0 38 154.3 45.6 3.4
0 0 40 55.8 27.8 2.0
0 0 41 35.5 23.7 1.5
0 0 43 -25.3 34.4 -0.7
0 0 44 67.7 32.1 2.1
The data has an overall completeness of 99.7%.
When I run the data trough xtriage, it tells me:
Statistics depending on twin laws
-----------------------------------------------------------------
| Operator | type | R obs. | Britton alpha | H alpha | ML alpha |
-----------------------------------------------------------------
| -h,-k,l | M | 0.307 | 0.301 | 0.256 | 0.022 |
-----------------------------------------------------------------
Patterson analyses
- Largest peak height : 5.288
(corresponding p value : 0.86552)
The largest off-origin peak in the Patterson function is 5.29% of the
height of the origin peak. No significant pseudotranslation is detected.
The results of the L-test indicate that the intensity statistics
are significantly different than is expected from good to reasonable,
untwinned data.
As there are twin laws possible given the crystal symmetry, twinning could
be the reason for the departure of the intensity statistics from normality.
It might be worthwhile carrying out refinement with a twin specific target function.
I also estimated the twin fraction with the following server:
http://nihserver.mbi.ucla.edu/Twinning/
And found it to about 20% (using data from 6-3A).
After phasing the data with rigid body refinement in Refmac with intensity based twin refinement (brings both R-factors to ~36%), I have tried doing a twin refinement with Phenix:
%phenix.refine model.pdb data.mtz twin_law="-h,-k,l" strategy=rigid_body+individual_sites+group_adp
The R_factors consistently diverge to the following values:
Final R-work = 0.3392, R-free = 0.4360
Simulated annealing/TLS/ordered solvent/messing with wxc/wxu weights does not seem to improve the situation. I have also tried this with and without prior detwinning with the CCP4 program "Detwin", with similar results.
Reindexing to -h,-k,l gives similar R-factors after rigid body refinement but after refinement with Phenix, things get even worse:
Final R-work = 0.3919, R-free = 0.5059
I was surprised to see that the fit to the map using both indexing conventions are comparable even though this should effectively reverse the hand of the structure. I assume this is because twin refinement is basically doing the job of "partial reindexing" on those parts of the lattice which are twinned?
I have been working on this for about a week now and can't for the life of me find a way forward. If anyone can offer any suggestions It would greatly be appreciated. Im slowly beginning to lose my sanity....
BTW I don't think my problems stem from misindexing, although the beam was wondering all over the place during this block of data collection, I have previously solved another structure at the same d and xbeam ybeam values without any trouble.
Sorry about the v.long post. Thought it would be better to give too much information rather than not enough =)
Many thanks for all your suggestions in advance and for everything I
have learned from this community in the past. Cheers,
Joe
PhD student.
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