[ccp4bb] Twinning problem

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: difference of R work and R free as a function of resolution
From: Dirk Kostrewa kostrewa {- at -} LMB {- dot -} UNI-MUENCHEN {- dot -} DE
Date: 2007-11-16

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Subject: SC-CYS-MET drop-out
From: "Wisecarver, Sarah N" swisecar {- at -} UTK {- dot -} EDU
Date: 2007-11-16

Subject: Twinning problem
From: Andrew Torelli andrew_torelli {- at -} URMC {- dot -} ROCHESTER {- dot -} EDU
Date: 2007-11-16

To the CCP4 community,

I have collected data from an RNA molecule that extends to 2.9 angstroms, exhibit mosaicity less than 0.9 degrees and generally show nice, round spots. The crystals look cubic and are not birefringent (suggesting a cubic lattice). However, the data index poorly with the best solutions being either I23 (a=b=c=141.68) or I422 (a=b=141.74; c=141.57). Predicting reflections using each of these indexing solutions appears to confirm each as a valid indexing solution. However regardless of which space group I select to process the data, the final result after scaling reveals relatively high Rsym values overall and for individual batches (10% - 20%). Also, a large wedge of data are required to achieve nearly 100% completeness (>60 degrees; if the lattice was truly cubic I would expect much less to be required for high completeness).

These discrepancies led a colleague to suggest twinning might be a problem. The UCLA twinning server (Yeates method) finds the following:

For the data processed as I422 in the partial twinning test: No merohedral twinning laws found for that space group
For the data processed as I23 in the partial twinning test: Twin fraction of 0.408
For the data processed as I23 in the perfect merohedral twinning test:
Resolution ; / 2
16.147 ; 1.99 (n = 404)
7.224 ; 1.57 (n = 404)
6.067 ; 1.40 (n = 404)
5.434 ; 1.33 (n = 404)
5.006 ; 1.37 (n = 404)
4.687 ; 1.44 (n = 404)
4.440 ; 1.35 (n = 404)
4.240 ; 1.34 (n = 404)
4.070 ; 1.88 (n = 404)
3.924 ; 1.54 (n = 404)
3.799 ; 1.61 (n = 404)
3.689 ; 2.03 (n = 404)
3.590 ; 1.74 (n = 404)
3.501 ; 2.00 (n = 404)
3.421 ; 2.08 (n = 404)
3.347 ; 2.17 (n = 404)
3.278 ; 2.15 (n = 404)
3.216 ; 1.42 (n = 404)
3.158 ; 1.58 (n = 404)
3.104 ; 1.69 (n = 404)
3.054 ; 1.59 (n = 404)
3.007 ; 1.48 (n = 404)
2.962 ; 1.80 (n = 404)
2.920 ; 5.42 (n = 404)

This is my first experience with twinning (hmmm...I feel like I'm being initiated), and I have several questions that I have not been able to answer yet from researching the literature or CCP4bb archives. I should mention that several data sets from several similar crystals all behave the same in terms of the difficulties in data reduction and even the apparent twinning fraction (the same to within a few %). I know the first advice will be to try new conditions, but I wonder if I can work with these data since I already collected data sets for several derivatives and also anomalous. Any advice or literature references are greatly appreciated to any or all of these questions:

1.) How should I go about assigning/identifying the correct space group? Does the apparent presence of merohedral twinning for the I23 processed data, but not I422, indicated that I do not have a cubic lattice?

2.) How is it possible that the data processed in the lower symmetry I422 space group are not also found to be twinned? I can't visualize how the same merohedrally twinned lattice could be described without conflict in the lower symmetry space group.

3.) I looked at the original T. Yeates paper in Meth. Enz. regarding twinning. There is an example of data from plastocyanin which are perfectly twinned. The reported plot of / squared as a function of resolution show a fluctuation around 1.5 that looks similar to the values I reported above as output from the perfect twinning test. How does one determine from those plots whether or not you have perfect merohedral twinning? Should I consider the average value, the lowest value, the distribution, or is my apparent partial twinning fraction sufficiently far from 50% to be sure that I don't?

4.) I tried running the perfect- and partial-merohedral detwinning scripts in CNS for the data processed as the I23 space group. The result of the perfect-merohedral detwinning script resulted in generally higher values of / squared, but it's not clear to me what that means or how it is possible to detwin perfect merohedral twinned data. After the partial-merohedral detwinning script however, the twinning fraction dropped to 17%. Is that informative with regards to what space group I'm dealing with or whether or not I have partial vs. perfect twinning?

5.) The last questions are about how to proceed with solving the structure. As I mentioned, I have collected data that I hope to use for MIR, potentially including anomalous. With a twinning fraction of 17% after detwinning, is it possible/appropriate to solve the structure by MIR or SIRAS (I'm guessing differences in the twinning will just diminish my signal to noise for finding the heavy atom peaks)? I also understand that it is possible to solve a perfectly merohedrally twinned data set by molecular replacement. I have a partial MR solution using the I23 data that appears to have unique phase information. I know there are several refinement programs that could be used for twinned data. Can anyone recommend one that handles RNA well?

Thank you very much for your time,
Best Regards,
-Andy Torelli

Andrew Torelli, M.S.
Ph.D. Candidate, Dept. of Biochemistry & Biophysics
University of Rochester School of Medicine & Dentistry
Box 712, 601 Elmwood Ave, Rochester, NY 14642
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: difference of R work and R free as a function of resolution
From: Dirk Kostrewa kostrewa {- at -} LMB {- dot -} UNI-MUENCHEN {- dot -} DE
Date: 2007-11-16 Next message:
Subject: SC-CYS-MET drop-out
From: "Wisecarver, Sarah N" swisecar {- at -} UTK {- dot -} EDU
Date: 2007-11-16