Hi, I'm refining a 2.88 Angstrom structure and am getting a difference of greater than 6% between the R-work and R-free. The best result (I think) is what I have from minimization alone: Start R-work = 0.3219, R-free = 0.3505 (no bulk solvent and anisotropic scale) Final R-work = 0.2424, R-free = 0.3174 (no bulk solvent and anisotropic scale) Start R-work = 0.3220, R-free = 0.3500 Final R-work = 0.2411, R-free = 0.3176 The results from simulated annealing are the following: Start R-work = 0.3219, R-free = 0.3505 (no bulk solvent and anisotropic scale) Final R-work = 0.2454, R-free = 0.3322 (no bulk solvent and anisotropic scale) Start R-work = 0.3220, R-free = 0.3500 Final R-work = 0.2403, R-free = 0.3333 I have also done the refinement with ncs but ncs is probably not appropriate for my structure. The final R-free ends up being higher (though the difference between R-free and R-work ends up being 6-7%) My PI (who is on vacation until next week) said that it was a problem if R-work was more than 0.06 lower than R-free. Any recommendations for me? Is it possible to reduce this difference? Is it worth it to rebuild the refined pdb if the R-work is this different from the R-free? My refinement script is: phenix.refine ss46_p3_cv.mtz ss46_rebuilt.pdb \ output.prefix=results/ss46_rebuilt_sa \ strategy=individual_sites+group_adp cif_link.params nag-nag.cif \ twin_law="h,-h-k,-l" \ simulated_annealing=true (the cif_link.params and nag-nag.cif files are made to handle the N-glycosylation sites, I think). Thanks for your help! -Sam
Hi Sam, to help with questions like yours, recently we have developed and implemented in PHENIX a new tool called POLYGON. See Acta Cryst. (2009). D65, 297-300 for details.
I'm refining a 2.88 Angstrom structure and am getting a difference of greater than 6% between the R-work and R-free. (...) The best result (I think) is what I have from minimization alone: Final R-work = 0.2411, R-free = 0.3176
Here is the distribution of Rwork and Rfree for "all" structures in PDB refined at resolutions between 2.78 and 2.98A, that is similar to yours, and "<<<" indicates where your "best result" is standing with respect to this distribution: r_work_pdb data_points=1411 min/max/mean= 0.1860 0.2870 0.2252 0.1860 0.1961 121 0.1961 0.2062 190 0.2062 0.2163 207 0.2163 0.2264 233 0.2264 0.2365 249 0.2365 0.2466 175 <<< your structure 0.2466 0.2567 109 0.2567 0.2668 67 0.2668 0.2769 42 0.2769 0.2870 18 r_free_pdb data_points=1411 min/max/mean= 0.1940 0.3400 0.2744 0.1940 0.2086 4 0.2086 0.2232 28 0.2232 0.2378 73 0.2378 0.2524 163 0.2524 0.2670 234 0.2670 0.2816 363 0.2816 0.2962 298 0.2962 0.3108 149 0.3108 0.3254 71 <<< your structure 0.3254 0.3400 28 It seems like your Rwork is reasonably good, and although there are more than 100 models with similar or worse Rfree, I believe you can push it below 30%. Things to try: - TLS. Use TLSMD to define TLS groups. - Use "optimize_wxc=true optimize_wxu=true" keywords. This may take longer to run. - Although 2.88A is pretty low for automatic water picking, I would still give it a try. - Torsion angle dynamics. - Try the four above with and without NCS. Let phenix.refine automatically defined NCS groups, but manually check is the automatic choice makes sense. Once done with trying and obtained the best result, use the following tools for validations: phenix.real_space_correlation phenix.polygon phenix.model_vs_data or phenix.validation where the three above are included along with the cool things from Molprobity team. You need a recent version of PHENIX for this with the new GUI: http://www.phenix-online.org/download/nightly_builds.cgi Please let me know if you have any questions!
The results from simulated annealing are the following:
Final R-work = 0.2403, R-free = 0.3333
Yes, both Rfree and Rfree-Rwork seem pretty large.
I have also done the refinement with ncs but ncs is probably not appropriate for my structure. The final R-free ends up being higher (though the difference between R-free and R-work ends up being 6-7%)
This may indicate that the NCS groups were not optimal or TLS is needed to NCS-restrain only residual B-factors, or you need to optimize the weights (see above). Once again, please let me know if you have any questions. Pavel.
On Sep 1, 2009, at 8:18 AM, Sam Stampfer wrote:
My PI (who is on vacation until next week) said that it was a problem if R-work was more than 0.06 lower than R-free. Any recommendations for me? Is it possible to reduce this difference? Is it worth it to rebuild the refined pdb if the R-work is this different from the R-free?
My refinement script is:
phenix.refine ss46_p3_cv.mtz ss46_rebuilt.pdb \ output.prefix=results/ss46_rebuilt_sa \ strategy=individual_sites+group_adp cif_link.params nag-nag.cif \ twin_law="h,-h-k,-l" \ simulated_annealing=true
Random ideas: - TLS (this sometimes has a huge effect on Rfree-Rwork) - Individual ADPs (may or may not help, but appropriate to try at that resolution) - What are your rms(bonds) and rms(angles)? If these are very high (or very low?) this may be the problem, and you need to optimize the weighting. - You should look at the other validation criteria as well (Ramachandran, rotamers, clashscore, etc.). -Nat ------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]
participants (3)
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Nathaniel Echols -
Pavel Afonine -
Sam Stampfer