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 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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Hi, from your description you seem to be doing the right things but the Rfree-Rwork gap is indeed rather large than expected at this resolution. If you send me model and data (off list) then there is a good chance I figure out what's happening and offer a  solution (if any ligand cif files involved -- send them too). Pavel On 5/24/19 08:51, Pham, Hang 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