[phenixbb] Twinning Refinement
Peter Zwart
phzwart at gmail.com
Thu Mar 25 22:22:58 PDT 2010
Hi Joe,
Some suggestions
1. Update your phenix version please. This is rather outdated.
2.0. If it doesn't refine in the normal cell / sg, toss this
preconceived notion away and:
2.1. Get your data in P1 (phenix.reflection_file_converter
data.sca--sca=p1.sca --expand_to_p1)
2.2. Do MR in P1
2.3 Assign free flags with phenix.refine (*IMPORTANT*)
2.4 Refine (a bit)
2.5 Run xtriage on the P1 data and provide the model as well (check
the phantastic GUI)
2.6 Check your RvsR stats to get an idea about the 'true' symmetry of the system
2.7 Read the RvsR paper quoted in the log file
2.8 Find out what this all means.
If confused about results, let me know.
HTH
Peter
2010/3/25 Joseph Brock <joeylives2ride at hotmail.com>:
> 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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>
--
-----------------------------------------------------------------
P.H. Zwart
Beamline Scientist
Berkeley Center for Structural Biology
Lawrence Berkeley National Laboratories
1 Cyclotron Road, Berkeley, CA-94703, USA
Cell: 510 289 9246
BCSB: http://bcsb.als.lbl.gov
PHENIX: http://www.phenix-online.org
CCTBX: http://cctbx.sf.net
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