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
>
>


-- 
=======================================================================
All Things Serve the Beam
=======================================================================
                                David J. Schuller
                                modern man in a post-modern world
                                MacCHESS, Cornell University
                                [log in to unmask]