Clemens hello
After not using SHARP for a wile as lesser tools were sufficient, we
start again
a demanding progect. To my very big surprise it appears that our
license (licenses)
have been expired
***************
***SHARP=ERR=+0005: Licence expired! ***
************************
it happened on biost7.tau.ac.il Mac server.
I also use my laptop with dynamic ID allocation to run SHARP. Is there
a method to check
the license status without actually running SHARP?
Please advise
Dr Felix Frolow
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel
Acta Crystallographica D, co-editor
e-mail: [EMAIL PROTECTED]
Tel: ++972 3640 8723
Fax: ++972 3640 9407
Cellular: ++972 547 459 608
On Nov 25, 2008, at 3:34 PM, Clemens Vonrhein wrote:
Hi,
9. The selenomethionine dataset was solved using MIRAS in SHARP/
autoSHARP.
The experimentally phased electron density yields contiguous
tracts of
density in the right place, unbiased density indicates a good
solution.
Model building was conducted in P212121, initially into the
experimental
maps and later with refinement in refmac using HL-coefficient-based
restraints. In some regions, sequence can easily be deduced from
clean
electron density (for the resolution). In other regions, side
chains are
missing, and in others, density is completely inconsistent with the
connectivity of the chains and highly conserved structural
elements. As
occurs sometimes with twinned data, many loops cannot be modelled
at all,
and the Rfree does not drop below 0.41 with an Rfactor of 0.34. The
result
is a model that is about 60-70% complete. Refinement was
performed with
and
without B-factor refinement.
It is not uncommon to have some severe density truncations in density
modification from difficult/poor data/phases. Since you have a partial
model, I would recommend a procedure we've used over the last years
quite
a few times: feed-back of the model into density modification (see
e.g. http://scripts.iucr.org/cgi-bin/paper?S090904950302394X). It is
easily done in the SHARP/Sushi interface:
- copy your latest/best model into sharpfiles/datafiles/model_xyz.pdb
- start a new density modification run with this initial model as an
additional phase information: our SOLOMON script will then start
not just from the SHARP phases, but from the SHARP+model
phases. There are some decisions you might need to do
* how many cycles to use this model: if you're worried about bias,
just use it for a single cycle. Usually, I would expect a model
that is based on some initial experimental map to be fairly
unbiased - unless you used some homologuous structure as an
initial model.
I usually use the model for say 10 cycles and doing 40 cycles of
density modification.
* you might want to redo the optimisation of the solvent content:
since you now start with a better initial map (hopefully), this
might give you a very different solvent content - initially poor
regions that were flattened might pop up.
- have a look at the resulting map (FBshasol/PHIBshasol column if you
don't use the tools in Sushi) and try to correct your model and -
maybe - complete it.
- go back to the start with a now improved model.
I tend to do this 3-4 times at least (sometimes not a lot seems to be
happening at the first 1-2 cycles, but often it suddenly picks up).
If you want to help your refinement (e.g. in REFMAC) with HLs from
SHARP: you want to use FP/SIGFP and HLA-D from the eden.mtz file (or
FPsha/SIGFPsha and HLA-D from a eden_flat_*.mtz file). These are in
synch with each other. Also: remember to build MSE residues and adjust
your f' value.
If you want to improve the HA refinement in SHARP you could use phase
information from a model or from a density modification run throughout
the HA refinement and residual map calculation (but _not_ the
phasing): if you're still missing a few Se or there are some alternate
conformations for the Se, this might be a good way of picking them up.
11. phenix.autobuild is able to build a polyalanine model that
covers
about
25% of the molecule.
I've had some very nice results recently with Kevin Cowtans BUCCANEER
(latest binary from his WWW page): it seems especially good in
building a lot of connected regions.
If you use such an automatic built model in the iterative procedure
mentioned above, you don't really have to worry about bias I guess.
Cheers
Clemens
--
***************************************************************
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
* Global Phasing Ltd.
* Sheraton House, Castle Park
* Cambridge CB3 0AX, UK
*--------------------------------------------------------------
* BUSTER Development Group (http://www.globalphasing.com)
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