Dear Joane, We have had very good luck refining low resolution structures with dramatic improvement using several newish options in refmac - particularly when NCS is present! Those options are jelly body restraints, automatic NCS restraints, and map sharpening. Here is a description cut-and-pasted from Garib's presentation (www.ccp4.ac.uk/schools/China-2011/tutorials/refmac_tutorial.pdf)
V) Low resolution refinement If you have older version of ccp4 then you can follow the instructions described in the presentation: www.ysbl.york.ac.uk/refmac/Presentations/Refmac_Erice_workshop.ppt slides 45-50 Full description of new features are in: www.ysbl.york.ac.uk/refmac/data/refmac_news.html In the new version of ccp4 there are options for jelly body, automatic NCS and map sharpening. a) Jelly body is under “Refinement Parameters”. You need to click “Use jelly- body refinement with sigma”. Change sigma to 0.01 or 0.02. This value defines “jelliness”. Smaller value means tighter restraints b) Automatic NCS restraints are under “Setup Non-Crystallographic Symmetry”. You need to cick “use automatically generated local NCS restraints”. You can also use global NCS c) Map sharpening is under “Monitoring and Output Options”. You need to click “Perform map sharpening with B value 20.0”. B value should a little bit smaller than Wilson’s B value. more in depth information can be found here: www.ccp4.ac.uk/schools/China-2011/talks/refmac_Shanghai.pdf This is definitely something you should explore. good luck, Eric ================================ Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu ================================ On Thu, 19 May 2011, David Schuller wrote: > This reminds me of: > > Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: Sequence > analysis and crystal structure determination > Barycki JJ, O'Brien LK, Birktoft JJ, Strauss AW, Banaszak LJ > Protein Science (Oct 1999) Vol 8, pp 2010-2018. > > In which the protein in question also had one monomer forming a dimer about a > crystallographic axis, and two monomers forming a dimer elsewhere in the > asymmetric unit. > > A portion of the molecule had messy density, which caused difficulties > literally for decades. The eventual solution was to switch to a different > species (human). After which, a MR solution of the original porcine data was > possible. I believe the disorder was judged to be intrinsic. > > > > On 05/19/11 09:02, Joane Kathelen Rustiguel wrote: >> Dear all >> >> >> I am refining a structure at 3.4 A resolution that contains 3 molecules in >> the >> a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric >> functional structure expected for this class of proteins. The other two >> chains >> B and C, which also form the functional dimer, seem to be, somehow, a lot >> more >> flexible than chain A. As a result, whereas the electron density map, >> b-factor >> and geometry for chain A is pretty reasonable for a 3.4 A resolution >> structure, the refinement for the other two chains (B and C) does not behave >> well. Even playing with different weights for geometry, analysing different >> levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map >> for the helical regions is ok, but the electron density map for strands and >> loops of chains B and C are broken along the main chain, B-factors are >> really >> high, and the geometry keeps being distorted. >> >> Right now, the R-factor and R-free are 24.2 and 28.6, respectively. >> >> Any suggestions in how to proceed the refinement? >> And even a more difficult question, how do we report this type of structure? >> How do we deposit those coordinates? We can certainly use chain A as a model >> to perform interesting studies of structure-function relationship, but we >> know >> that chain B and chain C have problems. >> >> Any help will be greatly appreciated. >> >> Regards >> >> Joane >> >> > > > -- > ======================================================================= > All Things Serve the Beam > ======================================================================= > David J. Schuller > modern man in a post-modern world > MacCHESS, Cornell University > schul...@cornell.edu >
