Joe, 1) The bulk solvent models in CNS and REFMAC are not stable at low resolution. Fixing the bulk solvent B-factor can improve this behavior.
2) With low resolution data model-bias becomes a serious issue when working with a MR solution. I have found that DM (with or withou NCS) can improve low resolution maps significantly. I routinely use RESOLVE to phase improve my model maps as I build new models (I use my PMB program to do this automatically for me using a combination of CNS/CCP4/RESOLVE http://xray.utmb.edu/PMB). In one low resolution MR case at 3.5A we had an entire domain in the wrong orientation and DM gave clear and unambiguous density for the correct orientation (caveat: 70% solvent does wonders for phase improvement). Good luck, Mark On Fri, 2007-10-19 at 11:39 -0400, 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 Yours sincerely, Mark A. White, Ph.D. Assistant Professor, Dept. Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Fax. (409) 747-4745 mailto://[EMAIL PROTECTED] http://xray.utmb.edu http://xray.utmb.edu/~white
