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Dear Iain Kerr,
this sounds very interesting. From what you say, it is quite obvious
that your true space group is I222 with probably one monomer in the
asymmetric unit (estimated) and that you have a clear molecular
replacement solution. You are, however, stuck with the refinement,
probably in some false local minimum. It is very difficult to make an
analysis from the distance, but I would like to point you to 1-2
potential pitfalls with TIM-barrels. Although your search model has 75%
identity, from my experience (with DHPS, a TIM-barrel), there are two
problems with TIM barrels. The first problem is that you have a kind of
pseudo-8-fold internal symmetry with TIM-barrels that might got you
stuck into an overall correct placement, but rotated by n/8 (n=1,7) with
respect to the true solution. The other problem lies in the variability
of TIM-barrels: by the time a tried to solve DHPS, David Banner (who
solved the very first TIM-barrel structure) analysed the PDB for
TIM-barrels, and found ~50 different entries, _all_ of which had
different relative arrangements of beta-strands and alpha-helices when
superimposed! Therefore, I would have two suggestions for you:
To get out of a potential false local "8-fold" minimum, I would try to
refine a a poly-ala model and see which side chain electron densities
come back. It could be advantagous, to reduce the resolution to 3 A,
first, to allow for a larger radius of convergence and then increase the
resolution again. You could also try torsion-angle simulated annealing
refinement with CNS. Maybe, this solves your problem already.
If this doesn't help, you could try several different related
TIM-barrels from the PDB as search models for MR. Maybe, you will get an
even better solution that helps you out.
This is all that comes to my mind at the moment.
Good luck,
Dirk.
Kerr, Iain wrote:
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I have a dataset that merges very nicely in C2 and I222 (Rmerges ~5% and ~8%
resp), 100% complete to 1.75A. The protein is a TIM-barrel enzyme, a couple of
loops are expected to be in different orientations or missing with respect to
the search model. The diffraction is very clean and appears to represent a
single lattice. The crystal is from a cluster with the same symmetry but which
presented multiple lattices upon diffraction...I just recently managed to
separate a single crystal and was very excited when I saw the diffraction at
the synchrotron....
The search model and target sequence have 75% identity and so I was expecting
this to be a routine MR problem. I have mutated the sequence of the search
model anyway to match the target sequence (I got tired of mutating the 25%
non-identical whilst fitting MR solutions..). Two loops are deleted (~30
resdiues). I find oustanding solutions easily in both spacegroups (no solutions
in I212121), using a number of programs including MOLREP, PHASER and CNS. None
of these refine (REFMAC5 and CNS). For the best solutions I have fit the model
to the density and the refinement does not improve (currently stuck at Rfree
~44%, Rfactor 40%). There are no obvious large errors excepting some missing
loops residues (~20 still). The best solution (MOLREP) shows some difference
density for one of the missing loops and waters and overall the maps are good.
I have checked for merohedral twinning (C2) and pseudo-translational NCS;
neither are present.
The indexing output from MOSFLM:
PENALTY
7 158 mP 55.55 82.57 82.27 90.1 109.9 109.5 P2,P21
6 12 oI 55.55 102.35 116.68 89.6 89.8 90.0 I222,I212121
5 10 mC 116.44 116.68 55.55 89.8 118.5 89.8 C2
4 8 mC 129.04 102.35 55.55 90.0 115.3 90.3 C2
3 5 mC 154.73 55.55 102.35 90.0 131.1 89.8 C2
2 2 aP 55.55 82.27 82.57 90.1 109.5 109.9 P1
1 0 aP 55.55 82.13 82.57 76.8 70.5 70.4 P1
I have tried solutions 6 and 4 (solution 4 is found by HKL2000 and I tried this first after I222). I have still to try 3 and 5 (3 looks non-standard with the large beta angle though, ..), which I just noticed when I re-indexed using MOSFLM today...at least with a similar crystal I have tried P1 and didn't get an MR solution.
In lieu of running another hundred MR jobs with multiple spacegroups I was
wondering if anyone had encountered anything like this before ?. Is there
something about C2 I am discounting ? I was expecting this problem to
disappear, having separated a single crystal from the cluster and therefore
removed the additional lattices from the diffraction pattern, but this is
apparently not the problem...
Thank you very much in advance for any help.
Cheers,
Iain
--
****************************************
Dirk Kostrewa
Paul Scherrer Institut
Biomolecular Research, OFLC/110
CH-5232 Villigen PSI, Switzerland
Phone: +41-56-310-4722
Fax: +41-56-310-5288
E-mail: [EMAIL PROTECTED]
http://sb.web.psi.ch
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