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
