You really cant refine B factors with 3.8A data ( or much else really!) Try fixing the B factor to that of the WILSON plot, and then do a few cycles of refinement to lok at the maps. You may see omitted side chains etc, but there is very little gain in much refinement. Best to try and collect more data..
Eleanor PS - I have never seen a better PHASER result! Yingjie Peng wrote: >*** For details on how to be removed from this list visit the *** >*** CCP4 home page http://www.ccp4.ac.uk *** > > >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] > > > > > >
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