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 ; <I2> / <I>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 <I squared> / <I>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 <I
squared> / <I>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
