On 08/18/10 08:25, Michael Thompson wrote:
Hello All,
I am currently solving a structure at 2A resolution with phases obtained from
molecular replacement. Using the MR solution, I began refinement with Refmac
using NCS restraints. I am currently building the parts of the model that were
left out of the MR search model, and have just about successfully completed all
three NCS-related chains. Obviously I will continue to use the most complete
model for refinement, and plan to release the NCS restraints over the parts of
the molecule that don't quite seem to obey perfectly.
My question is, once I have connected density for all three chains will it
still be worthwhile to perform density modification, such as solvent
flipping/flattening or histogram matching (implemented through SOLOMON and/or
DM) to improve phases? It seems that I have always been told that density
modification is typically carried out at the beginning of refinement, prior to
any model building, however my understanding of these types of density
modification (particularly solvent flipping/flattening) leads me to believe
that they would be most effective when more of the molecular envelope can be
identified, such as during later stages of refinement.
As building & refinement are a cyclical process, your density-modified
phases will keep improving as your improved model provides a
better-fitting mask. Once you reach the point where your model phases
are better than the density-modified phases, and improvement from the dm
maps is no longer apparent, you can quit the density modification. I
tend to continue the dm longer than some other people, because it is
free of some forms of model bias.
Also, I read something recently that lead me to believe that the solvent
flattening procedure may be implicit in the implementation of NCS averaging in
refinement software. I understand that the two processes are fundamentally
different and independent of one another, but the information I recently read
described something like the following (unless I misinterpreted). Because real
space NCS averaging requires identification of the molecular envelope in the
same fashion as solvent flattening, during NCS averaging the envelope is
identified then the map is solvent-flattened and averaged using the NCS
operator. I am unfamiliar with the inner-workings of most crystallographic
software, so I was wondering if this is how NCS averaging is implemented in
Refmac? I suppose another way to ask the question would be: If I have an
NCS-averaged map from Refmac, is it already solvent-flattened?
Different density modification software packages deal with the averaging
and solvent-flattening masks in different ways. Some packages
solvent-flatten everything outside the NCS mask. Having dealt with
situations in which the NCS relationship does not cover the entire
molecule, I do not like that, and prefer that NCS and solvent flattening
masks be dealt with separately (Schuller (1996) Acta Crystallogr. D52,
425-434.) IIRC, dm allows the option of dealing with the masks
separately and I cannot remember which behaviour is the default.
REFMAC is not a density modification application, but rather a
refinement application. NCS restraints may apply to only part of a
molecule if necessary.
Any help would be much appreciated. I am still relatively new to the refinement
process.
Thanks,
Mike Thompson
--
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All Things Serve the Beam
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David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu
