Hi Edwin, it's hard to tell without seeing the files. If you send me your most current model and data then I can have a look. Pavel On 7/24/15 14:13, Edwin Lazo wrote:
Hi all,
I am refining a structure that diffracted to 3.5 Angstroms and contains 6 monomers in the ASU. It is noteworthy to mention, in certain monomers, half their residues are missing (in other words no density). Initial b-factors after molrep were 118.585, R=0.32, and Rfree=0.40. Initial refinement was done with refmac and:
mean b-value=117.344 R=0.31 Rfree=0.40
Refinement with Phenix (phenix-installer-dev-2054-intel-linux-2.6-x86_64-centos5) was performed and the Ramachandran plot was bad:
OUTLIERS : 6.52 % ALLOWED : 13.67 % FAVORED : 79.82 % ROTAMER OUTLIERS : 7.18 % CBETA DEVIATIONS : 8
And final R=0.38 and Rfree=0.41.
I then improved Ramachandran plot in coot and follow Pavel's suggestion:
/- add H atoms: />>//>>/phenix.reduce model.pdb > model_H.pdb />>//>>/- then run a quick geometry regularization: *(**T**his part I **did in coot. phenix.**pdb**tools model_H.pdb --geom and phenix.pdbt**ools model_H.pdb --geometry**-**restraints do not work)* />>//>>/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 /My current stats are as follows: Final R=0.35Final Rfree=0.38ALL-ATOM CLASHSCORE : 173.74RAMACHANDRAN PLOT:OUTLIERS : 2.27 %ALLOWED : 4.61 %FAVORED : 93.13 %ROTAMER OUTLIERS : 36.26 %CBETA DEVIATIONS : 6Wilson B-Value: 108.4Mean B-value: 108.997I am unsure how to proceed from here. I am planning on removing some side chains which have no density and then refining with Rama. restraints. I can also send my mtz and pdb if that would help. The stats when I processed the data are as follows from xia2 -3d (xds, xscale): High resolution limit 3.50 15.65 3.50Low resolution limit 109.96 109.96 3.59Completeness 94.1 90.5 91.5Multiplicity 4.8 5.1 3.2I/sigma 11.3 26.5 2.4Rmerge 0.118 0.068 0.458Rmeas(I) 0.144 0.080 0.605Rmeas(I+/-) 0.144 0.083 0.584Rpim(I) 0.062 0.036 0.311Rpim(I+/-) 0.081 0.047 0.358CC half 0.992 0.994 0.791Wilson B factor 90.052Anomalous completeness 86.9 93.6 71.3Anomalous multiplicity 2.4 3.0 1.8Anomalous correlation -0.262 -0.440 -0.052Anomalous slope 1.005 0.000 0.000Total observations 169836 2234 7765Total unique 35071 442 2464Assuming spacegroup: P 21 21 21Unit cell:73.010 180.780 219.91090.000 90.000 90.000 Any help would greatly be appreciated, -- Edwin Lazo Scientific Associate Life Science and Biomedical Technology Research Resource National Synchrotron Light Source II Building 745 Upton, NY 11973 Office:(631)344-7247 Fax:(631)344-5059
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