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=0.35
Final Rfree=0.38
ALL-ATOM CLASHSCORE : 173.74
RAMACHANDRAN PLOT:
OUTLIERS : 2.27 %
ALLOWED : 4.61 %
FAVORED : 93.13 %
ROTAMER OUTLIERS : 36.26 %
CBETA DEVIATIONS : 6
Wilson B-Value: 108.4
Mean B-value: 108.997
I 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 3.50
Low resolution limit 109.96 109.96 3.59
Completeness 94.1 90.5 91.5
Multiplicity 4.8 5.1 3.2
I/sigma 11.3 26.5 2.4
Rmerge 0.118 0.068 0.458
Rmeas(I) 0.144 0.080 0.605
Rmeas(I+/-) 0.144 0.083 0.584
Rpim(I) 0.062 0.036 0.311
Rpim(I+/-) 0.081 0.047 0.358
CC half 0.992 0.994 0.791
Wilson B factor 90.052
Anomalous completeness 86.9 93.6 71.3
Anomalous multiplicity 2.4 3.0 1.8
Anomalous correlation -0.262 -0.440 -0.052
Anomalous slope 1.005 0.000 0.000
Total observations 169836 2234 7765
Total unique 35071 442 2464
Assuming spacegroup: P 21 21 21
Unit cell:
73.010 180.780 219.910
90.000 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
