[phenixbb] Twinning Refinement
Eric Larson
larsonet at u.washington.edu
Fri Mar 26 08:28:38 PDT 2010
Dear Joe,
It is not uncommon for proteins to crystallize in different crystal forms when in complex with different substrates or inhibitors. Trust that the absurdly high R factors (38% in the lowest resolution shell!?!?) despite the great lengths you went through with error models, etc. and the fact that scalepack is trying to throw out 20% of your data is a hint that you may be in the wrong spacegroup. Also, most of your "systematic absences" have an I/sig >2.5 and so probably are not absent. Were the predictions falling on spots during integration? This may provide another clue.
I think you need to go back to HKL and reprocess pretending that you don't know that the crystals are "well characterized". You may have a new crystal form.
good luck,
Eric
__________________________
Eric Larson, PhD
MSGPP Consortium
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195
> Date: Fri, 26 Mar 2010 05:13:07 +0000
> From: Joseph Brock <joeylives2ride at hotmail.com>
> To: <phenixbb at phenix-online.org>
> Subject: [phenixbb] Twinning Refinement
> Message-ID: <SNT120-W557FCF7E111C0FEA167769B230 at phx.gbl>
> Content-Type: text/plain; charset="iso-8859-1"
>
> 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 180? 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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