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

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On Wed, 23 Feb 2000, Susan Heffron wrote:

> 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?

I agree with Eleanor and Dirk that you're much better off using
constrained (strict) NCS at this resolution.  Dave is right that, in
theory, restraints with a high weight would do the job too, but the
programs tend not to deal with this properly because of numerical problems
introduced by having parameters with high curvature.  Refmac and TNT may
be more successful, when used in gradient/curvature mode, but I don't have
any experience.  So on the whole I would highly recommend using strict NCS
until you need to introduce restrained NCS.

> 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?

Dirk mentions using TNT, which has the advantage of allowing a B-factor
difference between NCS-related copies (an excellent idea that should be
emulated by the other programs as soon as possible).  It's true that the
official release of TNT does not contain a likelihood target, but we have
implemented the likelihood targets in a modified version of RFACTOR, which
we are willing to give to anyone who holds an academic license for TNT.

Randy J. Read
Department of Haematology
Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease
Cambridge Institute for Medical Research
Wellcome Trust/MRC Building
University of Cambridge         Tel: + 44 1223 336500
Hills Road                      Fax: + 44 1223 336827
Cambridge CB2 2XY, U.K.         E-mail: rjr27@cam.ac.uk