Dear bb users, I am refining a protein-ligand complex (at 1.68 A resolution) in which the ligand lies on a 2-fold crystallographic symmetry axis. The ligand occupancy is, therefore, 0.5 in each asymmetric unit.
I am almost at the end of the refinement but one problem has me stumped. Refmac keeps moving a carbon in the ligand too close to a serine OG and an oxygen too close to an arginine CD. Given that the ligand is at the interface, the density is not perfect. However, I rebuild the ligand to eliminate close contacts and still be within density and refmac pulls it right back close to the protein. The refined position does not even look better than the rebuilt one! It almost always looks worse! Would refmac put less weight on close contacts with the ligand because it is only partially occupied? I tried to use external restraints between the ligand and the residues so that they are kept further away. Upon searching the net, I found this command line: *external distance first chain [ch] residue [res] insertion [ins] - atom [n] [altcode [a]] second chain [ch] residue [res] insertion [ins]- atom [n] [altcode [a] ] value [v] sigma [s] [symm y/n] *I thought (hoped) that the distance herein is the minimum distance of approach between the specified atoms, I added these lines from within "Developer options" in refmac interface: exte dist first chain A resi 59 atom CD seco chain X resi 2001 atom O1 valu 3.2 sigm 0.02 symm Y exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu 3.2 sigm 0.02 symm Y It didn't recognize these restraints at all. However, when I change these lines to: exte dist first chain A resi 59 atom CA seco chain X resi 2001 atom O1 valu 3.2 sigm 0.02 symm Y exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu 3.2 sigm 0.02 symm Y Refmac recognizes the first line but not the second - lines from log file: Bond distance deviations from the ideal >10.000Sigma will be monitored A 59 ARG CA . - X 2001 DIE O1 . mod.= 5.024 id.= 3.200 dev= -1.824 sig.= 0.020 This raises two concerns: Concern 1: From the first line of output: the restraints here don't seem to be minimizing close contact at all; it seems to think they are bonded somehow (the distance between these atoms is not 5.024; it is 6.26 A; I don't know what 5.024 A is!). I am missing something here. It'd be great if someone can tell me what that is! Concern 2: This command only works when the first atom specified is a C-alpha atom (or maybe a main chain atom; I didn't try using other main chain atoms). Why is that? AND ULTIMATELY, is there some way I can tell refmac not to make the ligand and protein clash? I'd really appreciate any help! Thanks, Sangeetha.
