Hi, you might be able to distinguish the disulfide bonds from normal backbone when looking at the log-likelihood residual maps in SHARP: even for data collected at about 1A we often see anomalous peaks for sulfurs (and the data doesn't even have to be very good). And a S-S should be fairly strong.
If you want to improve those residual maps: you can easily include a partial model (e.g. poly-ALA backbone) into the heavy-atom refinement and phasing in SHARP. It often clears up the residual maps (to see your S-S and maybe Cys/met residues). You can also include the partial model during density modification (which uses SOLOMON) in SHARP/autoSHARP: this can help defining the first solvent envelope much better and giving you clearer density. If you have any slightly homologous model in the PDB: try phased molecular replacement (e.g. with MOLREP) to place this model. Looking at something with at least a similar fold might help you getting more confident with your directionality. Another thing you might want to try when looking at maps is the B-factor sharpening (see e.g. http://atb.slac.stanford.edu/public/papers.php?sendfile=194 for a paper). It is fairly trivial to use CCP4 programs to get the optimal B-factor according to that paper (let me know if you need help with this). Cheers Clemens On Fri, Jun 15, 2007 at 10:03:32AM -0700, Alexei Datsuk wrote: > Hi, > > I need some advice from experts. I have a low resolution electron density > map (3.7 angstroms) solved by SIRAS. The protein is a tetramer (47 > kDa/monomer) but I'm having trouble getting in register. There is very good > main chain density and clear secondary structure (beta-sheets) so I likely > have solved it correctly. I can trace most of the main chain as fragments > (loops connecting beta strands are broken) but I can't tell if I'm tracing > it in the right direction or whether I have it backwards. There is no real > density for side chains at all (except for Calpha), not even aromatics, so I > have no clue which way is N or C-terminus. There are a few disulfide bonds > too so I'm real worried about tracing incorrectly and never being able to > fix it later. My strategy right now is to build in as much poly-ala > fragments (I can get ~75% of trace as fragments) and then refine them. Then > redo phasing in SHARP with poly-ala model and SIRAS phases and then use DM > with NCS averaging to get improved map. Hopefully I can see then side chain > density. My question is how much bias would be incorporated if some of the > fragments are traced backwards? Would I be able to sort things out if some > fragments are traced backwards? Has anyone traced chains as fragments not > knowing which direction is which and then be able to sort things out later? > Right now I am stuck- I have spent the last 1 months trying to get things in > register but no luck. I don't want to build haphazardly but it doesn't look > like I have much choice. > > What have people done when tracing in low resolution maps? > > Thanks > > Alexei -- *************************************************************** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-------------------------------------------------------------- * BUSTER Development Group (http://www.globalphasing.com) ***************************************************************
