Hi -
With good 2.0 A data (as you seem to have) and a correct solution,
I would be rather surprised if ARP/wARP - REFMAC5 would not refine and
build a model without major trouble.
Did you try that at all ?
To be more detailed I would:
0. Put the mol rep solution in ARP/wARP and let it run (in the
meantime proceed to 1-3 if 0. fails ...)
2. Do some density modification using the model phases (if you are in
ccp4 simplest is to use DM)
3. Use the DM phases and pretend this is an 'experimental' solution in
ARP/wARP
More info and ideas are in:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18094467
Note that both ARP/wARP runs above can be done in the web server
without the need of local installation if you prefer.
http://cluster.embl-hamburg.de/ARPwARP/remote-http.html
Apologies for the shameless plug-ins but I really think its the
easiest thing to do, even if it might now work.
Tassos
On Feb 2, 2009, at 10:10, Nicholas M Glykos wrote:
Hi Xie,
Many thanks to all who responded to my earlier query (Drs. Paul
Swepston, Randy Read and Nicholas Glykos). I am trying to determine
the
structure of a very long coiled coil dimer (roughly 150 residues
long)
by molecular replacement. I don't know if it forms a canonical coiled
coil from end to end (it probably doesn't). I seem to get the right
solution using different lengths of polyala (and polygly and modeled
ones with side-chains) coiled coils as my search models (maps show
some
density for unmodeled parts of the coiled coil structure).
If your current best phase-set gives you back correct information
that was
not part of your model, you are doing fine. You are getting there
(but it
may take a while).
Starting from your current best polyAla model, you can give it a try
with
very many runs of torsion angle simulated annealing (starting from a
high
temperature), or with rigid-body simulated annealing (both from
within CNS
or XPLOR). Don't do positional refinement. With an almost perfect
polyAla
model you should expect R-free and R in the low 40s (%) range for ~2A
data.
However, building a model accounting for the entire sequence, getting
the correct chain direction and placing residue side-chains have
proved
difficult. And my R-facs have never improved beyond the low 50s
during
different refinement attempts. At the moment, I have 3 more or less
contiguous 30 residue stretches of coiled coil found by 3 independent
molecular replacement attempts using epmr (This was done to account
for
possible bends, stutters in the dimer). I am trying to get the rest
of
the model by new rounds of molecular replacement using epmr. I have
carried out exhaustive runs using epmr. But I haven't found the
rest of
the model. What are the options available to me with molecular
replacement? I have tried molrep, phaser and amore from the ccp4
suite.
My diffraction data is quite good (data between 25 and 2 A with an
Rsym
of 8%).
I'm not sure whether what you really need is more molecular
replacement.
Maybe you should invest more time with the maps (combined with 'safe'
simulated-annealing-based optimisation methods). What I've just said
only
applies if these 90 residues you have can indeed produce correct
information that was not part of the initial model (as you said).
Can I run Qs when a partial structure is available?
No. Sorry.
Nicholas
--
Dr Nicholas M. Glykos, Department of Molecular
Biology and Genetics, Democritus University of Thrace,
University Campus, 68100 Alexandroupolis, Greece, Fax +302551030613
Tel ++302551030620 (77620), http://www.mbg.duth.gr/~glykos/
P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
