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Re: [ccp4bb] problems with map quality, refinement and R/Rfree |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: problems with map quality, refinement and R/Rfree From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK Date: 2007-10-22 You must be carefulwith assigning twinning operators in a lower symmetry than the true one. Some twinning tests look for correlations between reflections which would be related by the twinning operator. eg a twinning operator for PG 4 is k,h,-l so the CC between h,k,l, and k,h,-l is checked. But if your true point group is 422 then that very operator is a SYMMETRY equivalent and of course you get near perfect correlation whether there is twinning or not. A safer indicattor of twinning is to look at the moments of acentric reflections - if the 4th moment of E is <<2.0 then you probably dO have twinning.. SFCHECK and Xtriage certainly use this and if they say there is no twinning I would be inclined to believe them! Eleanor James Holton wrote: > Merge in the lower symmetry space group you mentioned and refine with > a twinning operator. > xtriage can tell you what the twinning operator is and phenix.refine > can do the twinned refinement. > > In my experience, these kinds of stats almost always indicate an > incorrect symmetry choice somewhere (usually the space group choice, > but it can be twinning too). In general, it never hurts to re-merge > your data in P1 and then use PDBSET to expand your model into the P1 > cell. In cases like yours with centering you can use TRACER to tell > you what operators to use to get to a primitive cell. This should > never make your Rcryst any worse. If rigid body and/or further > refinement gives you better stats and significant difference features, > then you had the wrong symmetry. > > -James Holton > MAD Scientist > > Joe Smith wrote: >> Hi all, >> >> We have data sets for a protein-RNA complex at 3.2 A resolution. . The >> data belong to the space group I4122 and contains one molecule of >> Protein-RNA complex (300AA and 16nt; 66% solvent content) in the asym >> unit. We have managed to get phases using Se-SAD and the present model >> contains 60% of the protein atoms (250 residues many of them build as >> Ala) and almost complete RNA (16nt). We could not locate ~20 % of the >> residues (present in various loops as well as at N- and C-terminal >> ends). First 120 residues present at N-terminal end seem to have poor >> relatively main-chain density and almost no side chain density. RNA >> and 130 residues present at the C-terminal end have good electron >> density for main-chain as well as side chain. >> >> The solution seems to be correct as molecular replacement trials with >> this model gave same solution in Phaser, Molrep and AMoRe. Solution >> seems to be also correct because as expected one RNA strand present in >> asym unit pairs with another one coming from symmetry related >> molecule. >> >> We have checked data in space group I4122 with CCP4 (SFCHECK, >> cumulative intensity distribution), Yeates server as well as >> Phenix.xtriage. In all the cases it indicates no twinning. However if >> I process the data in lower symmetry space group it does indicate >> presence of almost perfect twin. We realize that this could be just >> because of processing data in lower symmetry space group and data in >> all probability may still be fine. >> >> The problem is now with refinement and model building. Refinement with >> CNS and Refamc gave considerably higher value of R and R free. In >> CNS1.1, refinement with small changes in the model some time leads to >> large shift in R and R-free value. >> Refmac: R=36; Rfree=47 >> CNS1.1: R=40-59; R-free=45-63 >> However in both the cases map looks more or less same (with reasonably >> good density for the main-chain as well as RNA) >> >> I tried refining with phenix.refine and there is some improvement in >> the R(34%) and Rfree(40%) values. I think may be robust bulk solvent >> correction incorporated in phenix.refine has helped in this case. >> However, I still see no improvement in the map quality for the first >> 120 residues. >> >> In the absence of any clear density I am unable to build any further. >> I feel N-terminal domain is bit flexible and may have overall poor >> density. I have used Se position as well as predicted secondary >> structure in assigning the amino acid in the map but due to few breaks >> in the N-terminal domain as well as poor density I am unable to assign >> any amino acid into the poly ala main chain. >> >> Frankly speaking, I do not know how to proceed further. I welcome any >> kind of suggestions which could help us in this case. >> >> Regards >> Joe >> > > CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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