Hi,

A high Rfree can indicate that the refinement strategy is not appropriate.

Several suggestions:

1) At 2.5 A resolution, you probably don't need reference model restraints. 

2 ) Simulated annealing (SA) is most useful early in refinement, when the model is far from convergence. Manually built models, or MR solutions involving significant local conformational changes, are common inputs where SA can improve over simple gradient-driven refinement. It is generally less helpful later in refinement, and/or at high resolution.

3) Individual B-factors and TLS are mutually exclusive, so did you define different strategies for different areas? If yes, you could try to revisit the parameterization. 

4) It might be also worthwhile to double check the secondary structure assignment for secondary structure restraints.

Weight optimization is typically done at the end of refinement. So while you are trying to figure out the strategy, you might want to disable it, as it is time-consuming.

Best wishes,

Dorothee

On Thu, May 23, 2019 at 5:54 PM Pham, Hang <Hang.Pham@iaf.inrs.ca> wrote:

Dear all,

I am refining my structures at 2.5 A. The model is well fitting in the electron density. I already add water and ligands. 

I’m trying these options (XYZ coordinates, real-space, TLS parameters, Individual B-factors, Optimize X-ray/stereochemistry weight, Optimize X-ray/ ADP weight, Reference model restraints, Use secondary structure restraints, Simulated annealing (Cartesian), Automatically correct N/Q/H errors) to refine the structure but the R-free still be high.

The reference model is 98% identity with the structure that I’m building. After refining, the R-work always decrease while R-free increase. My last values for R-work is 0.2501 and for  R-free is 0.3246.

I’m lost and don’t know what to try more.

Can anyone could explain me what happens here ? What others options that I could use to optimize R-free value ?

Thank you in advance !

Hang Pham

------------------------------------------------------
Hang Pham, Ph.D Student,
The Doucet Lab
Centre Armand-Frappier Santé Biotechnologie
Institut National de la Recherche Scientifique (INRS)
Université du Québec
Réseau International des Instituts Pasteur
531 Boulevard des Prairies
Laval (Québec) H7V 1B7  CANADA
Téléphone : (450) 687-5010, poste 4210
Site web: http://www.profs.inrs.ca/ndoucet/

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