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Hi Nick, > in a structure that i am currently working on via mol rep, i see difference density for a peptide ligand. when i build the ligand and do refinement in REFMAC 5 and look at the 2fo-fc map, I see nice density for > the ligand now. but, if I then use the coordinates with the ligand to look at a density modified map within CNS, i only see density for bits of > the ligand. does this mean that the 2fo-fc map is wrong or that the This turned out longer that I expected...the short version is to check the mask used in cns solvent flipping (may be assigning ligand region as solvent), try pirate, and read B.C. Wang's Methods in Enzymology paper. Longer version follows... Regarding density modification - are you using model phases or experimental phases as your original phase set? I'm personally cautious regarding density modification of model phases, although it seems to be a reasonably common practice. Another thing to keep in mind is that there are several different density modification schemes, and they can behave slightly differently. If I remember correctly, cns uses solvent flipping (solvent flattening with gamma correction, similar to dm). Your ligand density may be getting classified as solvent during mask generation (if you're generating your mask by spherical averaging, then this is a possibility): you could try changing a range of different sphere radii for mask generation and see how the map changes. If you are generating the mask from the protein model, then the ligand might be getting cutoff there as well (if this is the case, I don't have any suggestions for how to proceed without introducing bias into the maps...although if you're using a model-derived mask on model phases, I'd be skeptical that it would be of much use). With input experimental phases, manually setting part of the protein region to solvent weakens the density but doesn't eliminate it, so your suspicion regarding ligand disorder could be reasonable. I haven't tested this with model phases, so I don't know how this would behave. You could try pirate, which uses a density modification procedure that doesn't use a mask (my simplified understanding is that it's essentially maximum likelihood histogram matching, but this is probably an oversimplification). If you do try this, I'd recommend testing several different reference structures. When I was testing it, the solvent percentage of the reference structure seemed to have the largest effect on the final map, but this may vary with your data. As far as references go, B.C. Wang's Methods in Enzymology is a very clear introduction to solvent flattening, although it doesn't cover more recent developments (gamma-correction, histogram matching, etc). Good luck, Pete Pete Meyer Fu Lab BMCB grad student Cornell University
