And on a related note, how can one be sure that the FreeR set that phenix.refine is generating takes the given twinning operator into account? From the log file output, it seems like the FreeR set is generated before it gets to any of the twin statements. I have the following in my file, and I can see during the refinement that it is indeed doing twin refinement, however I want to ensure that it's using a reasonable FreeR set. twinning { twin_law = "-h,-k,l" detwin { map_types { aniso_correct = True } } } r_free_flags { fraction = 0.05 max_free = 5000 } Thanks! Chris ________________________________ From: [email protected] [mailto:[email protected]] On Behalf Of Christina Bourne Sent: Monday, May 19, 2008 1:32 PM To: [email protected] Subject: [phenixbb] problem with high Rfree Hello BB, I am working on a structure that is twinned (merohedral) in P41. Initial estimates of twinning fraction (via xtriage and Yeates server) are around 0.37; in refinement they start around 0.49 and drop to 0.46 (so far). My MR soln is good (Phaser LLG ~550) with 4 mols per AU; this gives good packing with no room for another mol. I have data from 48-2.8A. I should mention that data processing wasn't easy- this crystal form also has an apparent epitaxial twin in the b dimension causing a doubling in that dimension. During indexing (with SAINT) I applied a transformation of 100 00.50 001 to the reflections, which then gave me the expected cell and from Xprep the data looks OK. (Yes, I am trying to grow different forms) Maps look nice, although I wish my ligand had more/better density. I ran phenix.refine with defaults, and included twin_law="h,-k,-l", xray_data.r_free_flags.generate=true, detwin.map_types.aniso_correct=true and also simulated_annealing=true. My starting R/ Rfree is 0.448/ 0.4385; ending is 0.2962/ 0.4270. Geometry is somewhat tighter after refinement but is better (bonds 0.017 to 0.01, angles 1.966 to 1.530). I have not included my ligand, and I can see a few spots where the model needs adjusting, but nothing majorly wrong. Why is my Rfree hanging so high? Is it becaues I have bad karma? Is it to do with the twinning? I am pretty sure my space group is right (based on the above info and the maps look fine; Phaser could not find a solution in P43). There isn't room for anything else in the cell. Any opinions or suggestions are most welcome as I wait for better xtals to grow... Thanks in advance, Christina

Hi Christina, I usually observe similar R/Rfree behavior when I use LS target instead of maximum-likelihood. In fact, twin refinement in phenix.refine is based on LS target function. This is just to share my observations; it's not necessarily happening in your case. - How many of test reflections you have and how they are distributed in resolution bins? - Just out of curiosity: did you try to ignore twinning and run refinement with default target? - What if you don't run SA? - Did you try to play with target weights (try different values for wxc_scale and wxu_scale) to see how this affect the R-factors? - Are you using the latest (or one of) phenix.refine? - Did you try to fill in water? Could you send a log file: may be we could spot something outstanding... Pavel. On 5/19/2008 1:32 PM, Christina Bourne wrote:

Hello BB, I am working on a structure that is twinned (merohedral) in P41. Initial estimates of twinning fraction (via xtriage and Yeates server) are around 0.37; in refinement they start around 0.49 and drop to 0.46 (so far). My MR soln is good (Phaser LLG ~550) with 4 mols per AU; this gives good packing with no room for another mol. I have data from 48-2.8A. I should mention that data processing wasn't easy- this crystal form also has an apparent epitaxial twin in the b dimension causing a doubling in that dimension. During indexing (with SAINT) I applied a transformation of 100 00.50 001 to the reflections, which then gave me the expected cell and from Xprep the data looks OK. (Yes, I am trying to grow different forms) Maps look nice, although I wish my ligand had more/better density.

I ran phenix.refine with defaults, and included twin_law="h,-k,-l", xray_data.r_free_flags.generate=true, detwin.map_types.aniso_correct=true and also simulated_annealing=true. My starting R/ Rfree is 0.448/ 0.4385; ending is 0.2962/ 0.4270. Geometry is somewhat tighter after refinement but is better (bonds 0.017 to 0.01, angles 1.966 to 1.530). I have not included my ligand, and I can see a few spots where the model needs adjusting, but nothing majorly wrong.

Why is my Rfree hanging so high? Is it becaues I have bad karma? Is it to do with the twinning? I am pretty sure my space group is right (based on the above info and the maps look fine; Phaser could not find a solution in P43). There isn't room for anything else in the cell.

Any opinions or suggestions are most welcome as I wait for better xtals to grow... Thanks in advance, Christina

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