I had a similar issue (8 chains in a large ASU, 2.4 A resolution, 29%
homology, possibly--and ultimately confirmed--twinned data). Here is how
I solved my structure (not pretty!):
1. Phaser was used to find an initial placement for the chains using
(what turned out to be) the twin maps. This actually worked fine to
get a grossly correct position for all chains in the ASU. But the
low homology structures were pretty much useless as a starting point
for refinement in Refmac, and the twin maps were, um, just bad, as
you would expect. I used this structure as a starting point for
phases for the next step.
2. Once I had that, is used Parrot and Buccaneer to do density
modification with NCS and autotraced about 98% of the structure,
with only a couple of gaps/insertions.
3. Running Phaser with the twin option enabled identified the twin
fraction easily and resulted in excellent maps for further refinement.
If I had been quicker in recognizing the twinning of the data, I
probably could have detwinned the data from the start, and Phaser,
perhaps in combination with Parrot & Buccaneer with NCS, would have
arrived at a solution quicker.
If you have not used Parrot with NCS pipelined with Buccaneer, give it a
try. I was very pleasantly surprised (and impressed) at how efficient
this was at getting me to a place where I could start refining the final
structure.
Your data quality may hamper your ability to solve the structure,
especially if you have a large number of overlaps in your
high-resolution data. In that case, I wonder if you might fare better by
using data cut off to a lower resolution, where the overlap issue is not
as severe. With your unit cell, you are probably looking at a long
camera distance and thin-slicing to get usable data. That's what we have
to do with a 50 x 150 x 250 A unit cell (C2) which is favored by one of
our favorite proteins and its variants.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 10/26/2012 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.
========================================
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
========================================
