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Re: [ccp4bb]: low res dataset, need help with NCS constraints

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Dear Susan Heffron,

I believe (yes, believe), that at this resolution the safest way to refine
your model is by applying NCS constraints, which really increases your ratio
of reflections to atomic x,y,z parameters to 12000:9750. To my knowledge,
the only refinement program capable of defining (or leaving out) NCS
constraints for different domains is TNT 5E. (The next release, TNT 5F, even
allows a B-factor shift between different copies, which is VERY useful.)
However, TNT uses the least-squares target, rather than the
maximum-likelihood target, and its bulk solvent correction is done by
double-exponential scaling (REFMAC uses the same technique), which performs
less spectacular than the mask bulk solvent correction used for instance in
CNS. If you want to combine TNT's NCS constraints but with a
maximum-likelihood target and a mask bulk solvent correction, I would
recommend to contact Gerard Bricogne to run BUSTER/TNT for you (if you are
interested, I can give you his e-mail address). Otherwise, I agree with Dave
Schuller - the probably easiest way would be to run CNS with arbitrarily
high weights on the NCS restraints for your domains.
Please, beware: all NCS constraints/restrains are applied on your model and
help to stabilize the refinement. For your actual electron density maps you
have the choice either to calculate the usual maps from your model with NCS
(which should be better than without NCS due to less overfitting), or, if
the quality of your maps is not good enough, to do addtional real space
averaging on these maps which will again increase the signal-to-noise ratio,
but you will loose information about differences.

Good luck,
Dirk Kostrewa

Susan Heffron wrote:

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> ***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***
> Hi all!
> I could use some advice about refining a model with some fairly low-
> resolution data.  Here's my situation:
> I have a relatively low resolution dataset (~3.2A) in P212121, which
> consists of about 12,000 reflections.  My model is a molecular
> replacement solution, with two molecules in the a.u. and a total of
> about 6500 atoms.  I have been having trouble getting the refinement
> (using CNS) to proceed.  My R-free is still above 30%.  One concern I
> have is that the data-to-parameters ratio is less than 1 (12,000
> reflections, compared to 19,500 x-y-z positions).  I have been using NCS
> restraints, but wonder if constraints would be better, in order to
> improve the data-to-parameter ratio.  Am I correct in thinking about it
> this way?
> If I do use NCS constraints, I have the complication that my protein is
> a multi-domain molecule that has flexibility in the orientations of the
> domains to each other. So, it would have to be constrained
> domain-by-domain.  Which programs can handle this?
> One other possibility I've considered is to restrain the structure to a
> higher resolution copy with harmonic restraints.  This would require
> superimposing a higher-res structure, again domain-by-domain, and then
> refitting any loops that are obviously different in the current maps.
> (My current structure has a ligand bound, which makes the domain
> positions differ from other previous crystal structures even more than
> the normal flexibility.) Has anyone had experience with this type of
> situation to know whether it would be worthwhile to go through this type
> of rebuilding procedure and then restrain it with harmonic restraints
> during refinement?
> Thanks for any suggestions.
>                                Susan Heffron
> --
> ------------------------------------
> Susan Heffron
> Dept. of Physiology & Biophysics
> University of California, Irvine
> Irvine, CA  92697  U.S.A.
> ------------------------------------


* Dirk Kostrewa            e-mail: dirk.kostrewa@psi.ch *
* Paul Scherrer Institut   tel: +41-56-310-4722         *
* Life Sciences OSRA/007   fax: +41-56-310-4556         *
* CH-5232 Villigen PSI, Switzerland                     *