Hello Jose,
Depending on what data integration program you used, trying XDS may help
you out a little with spot overlap.
Example #3 in my rather out-of-date page:
http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html
illustrates how you could find 8 domains, especially if you pay
attention to the rotation angle values for the candidate domain
solutions. This example did not have twinning but did have a little
pseudo-centering. This is a 15 year-old example from back when I was
using AMORE, so I should clearly rewrite that page.
Additionally, if the inter-domain flexibility is restricted to rotation
about a single axis, it would be a good idea to rotate your model so
that this rotation axis is parallel to the Z axis. This was a method
that was exploited with Fab structures (whose elbow angle is a fairly
restricted rotation). If so oriented, rotation function peaks relating
different domains in the same molecule should show very similar alpha,
beta and differ in gamma.
Good luck,
Phil Jeffrey
Princeton
On 10/26/12 8:27 AM, Seijo, Jose A. Cuesta wrote:
Hi all,
I am dealing with a molecular replacement problem for a 60KDa protein
composed of 2 rigid domains joined by a flexible linker which can move
relative to each other. Sequence identity for my best model is 46%
evenly spread, so in principle this should be a tractable problem.
Then the problems start to pile up:
a)The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21.
Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in
P222 with the same a, c and b axes, now of course in that order. In the
P21 case, that corresponds to 4 monomers in the asymmetric unit with a
solvent content of approx. 50%, giving me 8 domains to find if I
separate them.
b)The 288 axis means that my data show some overlap in almost all
orientations (might be corrected in the future with new datasets), so
that my low resolution data are likely unreliable.
c)Intensity distributions suggest twinning in either point groups.
Actually, they are beyond the perfect twinning case, which I attribute
to the reflection overlaps making the strong reflections weaker
(integration box too small) and the small stronger (from tails of
adjacent strong ones). Of course the latest would mean that the twin
fraction estimation is unreliable, but all moments, etc show perfect
twin statistics, so I am assuming that there is indeed perfect twinning
of some sort.
So, the question is, what is the best strategy to deal with this many (4
or 8) body / noisy / twinned problem?
I am trying EPMR with many bodies, but I suspect the twinning would
throw it out of the right track, and one domain seems to be too little
of the diffracting matter to show any sort of discriminations between
solutions and non-solutions if do the usual serial searches. I plan to
let autotracing programs be the judge of success, but I am not sure of
how well those can deal with twinning. Can Arp-Warp use twinned data?
Thanks in advance for any tips.
Jose.
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Jose Antonio Cuesta-Seijo, PhD
Carlsberg Laboratory
Gamle Carlsberg Vej 10
DK-1799 Copenhagen V
Denmark
Tlf +45 3327 5332
Email josea.cuesta.se...@carlsberglab.dk
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