Re: [ccp4bb]: high resolution refinement

[Victor Lamzin <Victor@embl-hamburg.de>]

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Jan Abendroth wrote:
> 
> I would like to pose one question concering refinement of
> high-resolution structures. This topic has been discussed to some extend
> at the beginning of this year, but I would like to go for sure and have
> an additional question:
> 
> The situation:
> I have refined a quite large structure (6 monomers of 50kDa each in the
> asu) at 1.7AA to quite reasonable R-values (Rwork: 16%, Rfree: 19%). Now
> I have measured a 1.295A data set of the same crystal. The unit cell
> dimensions differ less than 1%. Because of the huge amount of data
> (close to 700.000 independent hkls) refmac5 and ARP/wARP with refmac4
> appear to be THE refinement-programs to use (also in terms of tolerable
> time for refinement).
> 
> The questions:
> Is it the reasonable way to start the refinement of the 1.295AA
> structure right from the beginning with all the data I have and let
> maximum likelihood find its way. Or is it better to use the "old
> fashioned" way to extend the high resolution limit in small steps and
> search water in each step?

Dear Jan,

I suggest to use all data straight away. Since your cell parameters
slightly differ and the position of the molecule may be affected,
the quickest would be either:

Run molecular replacement

or 

Run PDBSET/COORCONV to convert crds to frcs and then back to orths with
the new cell parameters.

> Is the following refinement strategy reasonable (each step until Rfree
> converges):
> - refinement at 1.295AA without water starting from 1.7AA structure with
> refmac5
> - adding water with ARP at 1.295AA
> - refinement with anisotropic B-factors
> - introduction of multiple conformations
> - refinement of occupancies
> - refinement with hydrogen contributions

In our experience it is quicker to start with addition of solvent sites
at 1.3 A (refinement of both bulk solvent and overall anisotropy within
REFMAC), followed by introduction of riding hydrogens, then anisotropic 
refinement and finishing up with multiple conformations and refinement 
or estimation of their occupancies.

> Taking in account resolution and the amount of data: Where should the
> R-values converge after proper refinement?

Very much depends on the Wilson plot B factor and overall anisotropy.
A typical value for R factor would be 11-13 %.

Regards, Victor