Hi Teresa,
As Eleanor has mentioned, you should probably check out other space
groups. Xtriage gives a lot of great information and many plots to
inspect. But, if you do not know what the plots mean and just look at
the results that say the twin fraction is 0.48 you can get into some
trouble. From what I have seen over the years, when most people say they
have a twin fraction nearly 0.5, they are usually in the wrong space
group and the data is not twinned. When the summary table shows a low 'R
obs' and a high twin fraction across the tests that means that the
operator is present in the data. But that could be a crystallographic
operator or a twin operator. You would have to inspect the plots to see
if the data looks twinned, but if there is pseudosymmetry it could make
the plots look not twinned. You could run the extra twin tests in
Xtriage inputting the MR solution so that the R vs. R plot (Andrey
Lebedev, et al. (2006) , Acta Crystallogr. D62, 83-95) could be
calculated. In the end, your data could be nearly perfectly twinned, I
have had it happen a couple of times, but I always tell people to assume
it is not until the model building is done and the R-factor still won't
drop. Your R-factors are not that bad for 3A resolution, indicating the
operator might be crystallographic and you are too low in symmetry.
So if you have done all this and you still believe the data is
twinned... Don't detwin your data! Your twin fraction is too high and it
will introduce a lot of errors. This has been gone over many times and
you can search the archives. I never detwin data regardless of twin
fraction. Modern programs like Phenix.refine and Refmac (I think) handle
twinned data just fine. If you are just trying to calculate an ADF map
to find the Br then it might be difficult since the peak heights are
going to be lower. Your best bet is to increase your redundancy without
killing the crystal with too much radiation damage. If your redundancy
is around 10 or so then you could probably use the 'scale anomalous'
option. I tend to scale with and without it and look at the maps to see
if it helps or not. Sometimes it helps, other times it doesn't and I am
sure there are reasons (like anisotropy). Another word of caution is the
possibility of model bias in the maps when the twin fraction approaches
0.5. The programs will use the model in the detwinning process if the
twin fraction is above a certain threshold. I believe it is 0.45 in
Phenix.refine.
First thing I would do is follow Eleanor's lead and check out other
space groups. You might have pseudo-symmetry with or without twinning.
Jon
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist, NE-CAT
Argonne National Laboratory, 436E
9700 S. Cass Ave.
Argonne, IL 60439
Email: schue...@anl.gov
Tel: (630) 252-0682
On 03/13/2014 03:41 PM, Teresa Swanson wrote:
Dear collegues,
I'm working with a drug complexed protein structure that is having major
twinning issues. The drug has a single Br atom on a benzene ring, which I'd
like to use for orienting the drug in the binding site. I have various
anomalous data sets, ranging from 3.0A resolution, all scaled into P222 with a
Rlin of .125.
Using MR, the twin law (k, h, -l) and NCS restraints, I can confidently solve
the structure without anomalous, and the drug density is clear in the Fo-Fc
map, with Rw/Rf at ~.26/.29 and a space group of P21221. It might be important
to note that any simulated annealing I've tried invariably increases the Rfree
by 2-3%, so I've scraped it. As you can imagine, when using the twinned data,
the anomalous maps are weak and random.
I've used the Phenix "detwin" option in Xtriage to see if I can pull the anomalous signal out of
it. If I use the .mtz file that is output for MR and calculate the anomalous maps, it looks promising. The
twin fraction for the one particular dataset I've been using is estimated at approx .48. Is this too close to
50% to do the detwinning? Now I'm wondering how to properly refine this further. I'm assuming that since I've
"detwinned" the data, I do refinement without the twin law. But that gives an initial Rf of .38
when using it gives .31. Since I've already solved the structure without using the anomalous flag, can I
just use the "detwinned" reflections and the refined structure to calculate an anomalous map
(without having to redo the refinement)?
Mainly, my main question is about how to tease out and properly refine the anomalous data from a
twinned structure. Also, how much of a difference will it make to scale into P222 versus P21212.
And, if I have quite high redundancy, should I "scale anomalous" in HKL2000 or just use
the "anomalous" flag?
Any help on refining this twinned structure would be greatly appreciated!
Thanks,
Teresa
PhD Student
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist, NE-CAT
Argonne National Laboratory, 436E
9700 S. Cass Ave.
Argonne, IL 60439
Email: schue...@anl.gov
Tel: (630) 252-0682
