On Tue, Jun 19, 2012 at 1:17 AM, Qian Sarah wrote:

Hi, this may not be a new problem, but I tried every stretagy that I know from bb, but the geometry is still very bad. So I wish I can get some help from here. Crystal information:  SAD data. Resolution 3.0.  There is no anisotropy in the data.  R/Rfree : 22.7/26.8 (Rfree-flag is always from the Refine_data.mtz file generated after the first refinement.)    RMS(bonds):0.02    RMS(angles): 1.453    Ramachandran outliers: 3.0%    Ramachandran favored: 91.2%    Rotamer outliers: 18.7% !!    Clashscore: 31.84    overall score: 3.47    I tried different strategies, like autobuild and then refine; set wxu/wxc=0.3/0.3; turn on "Ramachandran restrains", "fix bad sidechain romaters"etc, however the paramters are still very bad, especially the rotamer outliers. Then I rebuild them manually in coot,after refinement the parameters are even worse.

A few suggestions: 1. Don't use Ramachandran restraints in Phenix - please! 2. Do use Ramachandran restraints when rebuilding in Coot - fix the problems manually, perform local real-space refinement, and check that the result makes sense. Ensuring that the geometry is as good as possible before you run another round of refinement will make Phenix work better. 3. Don't adjust the weights manually; use the automatic weight optimization. It takes a long time to run (unless you have lots of CPU cores), but it's going to save you time in the end. My guess is this is going to help with some of the 4. At this resolution the rotamer fitting in phenix.refine is unlikely to work very well. You need to fix these problems manually too, I'm afraid. If you're willing to send us the model and data (off-list), we may have additional suggestions.

p.s. phenix version: phenix-1.7.3-dev-1046.

You should update to the latest official release. -Nat

Hi Sarah, thanks for sending me the data. I spent 15 minutes trying a few things and here is the statistics I get: r_work = 0.2454 r_free = 0.2700 MOLPROBITY STATISTICS. ALL-ATOM CLASHSCORE : 13.60 RAMACHANDRAN PLOT: OUTLIERS : 0.89 % ALLOWED : 5.33 % FAVORED : 93.78 % ROTAMER OUTLIERS : 8.64 % CBETA DEVIATIONS : 0 which isn't that bad compared to the starting point: r_work = 0.2469 r_free = 0.2613 MOLPROBITY STATISTICS. ALL-ATOM CLASHSCORE : 48.87 RAMACHANDRAN PLOT: OUTLIERS : 2.49 % ALLOWED : 5.33 % FAVORED : 92.18 % ROTAMER OUTLIERS : 29.47 % CBETA DEVIATIONS : 10 Here is what I did (it's just one of possible ways of doing this; other scenarios may works as good as this one or better!): - add H atoms: phenix.reduce model.pdb > model_H.pdb - then run a quick geometry regularization: phenix.pdbtools model_H.pdb --geom - then strip off H: phenix.reduce -trim model_H_regularized.pdb > start.pdb - then run refinement using NCS and Ramachandran plot restraints: phenix.refine start.pdb def_data.mtz main.ncs=true --overwrite output.prefix=def2 main.number_of_mac=5 ramachandran_restraints=true rama_potential=emsley strategy=individual_sites+individual_adp - then take outcome of previous refinement and run the same refinement plus weights optimization: phenix.refine def2_001.pdb def_data.mtz main.ncs=true --overwrite output.prefix=def3 main.number_of_mac=5 ramachandran_restraints=true rama_potential=emsley strategy=individual_sites+individual_adp optimize_xyz=true nproc=6 Also, looking at the original maps I see a lot of places where simple fixing in Coot is just obvious, for both - rotamers and Ramachandran outliers. So you should do it first of all. Also, use Ramachandran restraints ONLY to preserve good starting model and NOT to fix problems. All in all, geometry problems you were concerned about are all easily fixable. One of the reason why these problems arise is that the map is really looking like 3A resolution map, may be even worse; plus there are places in the model that you need to fix manually (refinement would not be able to fix them). I will send you the files off-list. Pavel On 6/19/12 1:17 AM, Qian Sarah wrote:

Hi, this may not be a new problem, but I tried every stretagy that I know from bb, but the geometry is still very bad. So I wish I can get some help from here. Crystal information: SAD data. Resolution 3.0. There is no anisotropy in the data. R/Rfree : 22.7/26.8 (Rfree-flag is always from the Refine_data.mtz file generated after the first refinement.) RMS(bonds):0.02 RMS(angles): 1.453 Ramachandran outliers: 3.0% Ramachandran favored: 91.2% Rotamer outliers: 18.7% !! Clashscore: 31.84 overall score: 3.47 I tried different strategies, like autobuild and then refine; set wxu/wxc=0.3/0.3; turn on "Ramachandran restrains", "fix bad sidechain romaters"etc, however the paramters are still very bad, especially the rotamer outliers. Then I rebuild them manually in coot,after refinement the parameters are even worse. p.s. phenix version: phenix-1.7.3-dev-1046. Thank you very much! best, Sarah