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Dear all,
I have collected one 3.8A dataset. The dataset was indexed and scaled as P222,
determined as P212121 using PHASER because the MR using P212121 gave most
significant
result. The cell parameters are a=59.535, b=107.003, c=153.554.
The PHASER gave me a good MR result (as following).
*******************************************************
PHASER result:
Fast Translation Function Table: Space Group P 21 21 21
-------------------------------------------------------
#SET #TRIAL Top (Z) Second (Z) Third (Z) Ensemble
1 1 2010.62 (35.68) - - - -
Refinement Table: Space Group P 21 21 21
----------------------------------------
#+ = input number #* = output number
Unsorted (refinement order) Sorted in LL-gain order
Initial Refined Initial Refined
#+ LL-gain LL-gain Unique #* #+ LL-gain LL-gain Unique =#*
=#+
1 2139.13 2139.13 YES 1 1 2139.13 2139.13 YES
*******************************************************
But when I tried to do refinement, I felt confused. I have tried both Refmac5 in
CCP4 and CNS to do refinement. Some results are listed below.
*******************************************************
Refmac5 in CCP4i result:
1.
rigid body refinement using no prior phase information input;
using the pdb file from PHASER as input pdb file;
resolution range: 50.00-3.81;
use matrix scaling; diagonal weighting term 0.001 (or 0.5);
refine overall B-factor (or not)
give similar results:
R=0.356, Rfree=0.368;
Mean B value (overall)=69.0, with most B fator normal, but
extremely high (>120, even upto 200) for some region.
2.
restrained refinement using no prior phase information input;
using the rigid body refined pdb file from Refmac5 as input pdb file;
resolution range: 47.14-3.81;
use matrix scaling; diagonal weighting term 0.001;
refine overall temperature factors: yielding R=0.37, Rfree=0.39;
refine isotropic temperature factors: yielding R=0.39, Rfree=0.42;
refine anisotropic temperature factors: yielding R=0.38, Rfree=0.41;
refine mixed temperature factors: yielding R=0.39, Rfree=0.42;
*******************************************************
CNS result:
using the pdb file from PHASER as input pdb file;
after rigid body refinement, the R=0.40, Rfree=0.40, using initial B-factor
correction and bulk solvent correction, yielding following information,
B-factor correction applied to coordinate array B: -66.713
solvent: density level= 0.447787 e/A^3, B-factor= 146.09 A^2
after minimizing, using initial B-factor correction and bulk solvent correction,
the R=0.35, Rfree=0.48, with following information,
B-factor correction applied to coordinate array B: 4.669
bulk solvent: density level= 0.454096 e/A^3, B-factor= 182.182 A^2
I have tried different protocols. Minimizing without initial B-factor correction
but with bulk solvent correction did NOT make big difference, but minimizing
without initial B-factor correction and also without bulk solvent correction
yielding a higher R (0.37) but a lower Rfree (0.46).
*******************************************************
I am wondering the initial B-factor correction and bulk solvent correction in
CNS play what kind of role in the refinement. Can these corrections be applied
to low resolution dataset, like my 3.8A dataset? If yes, how to use them
correctly?
Thank you in advance.
Best wishes,
Yingjie Peng
Yingjie PENG, Ph.D. student
Structural Biology Group
Shanghai Institute of Biochemistry and Cell Biology (SIBCB)
Shanghai Institute of Biological Sciences (SIBS)
Chinese Academy of Sciences (CAS)
320 Yue Yang Road, Shanghai 200031
P. R. China
86-21-54921217
Email: [EMAIL PROTECTED]
