Only two comments:
a. At that resolution, constrained group occupancy refinement should work reasonably well (provided you can model the 2 entities). Then you also do not have clashes between the molecules, because Occ(A)+Occ(B)=1, meaning when one (A) is there, the other one (B) is not. This works with refmac (external keyword file); if you need more sophisticated occupancy re/constraints SHELXL may offer more opportunities. b. There is no necessity for the two NCS copies of the binding site to look exactly the same (non-equivalent). Maybe there is a good reason/story (accessibility, contacts etc) for one site to be occupied differently than the other one. Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Bratkowski Sent: Wednesday, December 13, 2017 11:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Overlapping ligand electron density Hello all, I am working on a ligand binds near the active site of the protein, such that part of the ligand would clash with part of the natural substrate. I recently co-crystallized the enzyme with both molecules and solved the crystal structure to high resolution (around 1.4 angstrom). Surprisingly, the structure appears to contain both molecules. A few atoms from both molecules are located only ~1.4 A apart and are clashing (although not overlapping). The electron density between them looks connected, but based on the two groups that are clashing (a methyl group and a carbonyl oxygen), I do not think that a covalent adduct occurs. I had a few questions. 1) My guess is that the crystal is "sampling" two different conformational states and that both are visible due to the high diffraction resolution. The substrate contains a ring that shows a characteristic "hole" in the electron density and binds in the exact substrate binding site, suggesting that it is not a different molecule (no molecules with ring structures were included in the sample, crystallization buffer, or cry-protectant). One of the two proteins in the ASU contains electron density for whole substrate, while the other site has only density around the ring. However, a sizable amount of red FoFc density is present around the substrate, suggesting that it is only partially occupied. Does this explanation seem plausible? 2) How would I go about modeling these two molecules in the structure? Should I include both molecules (in their entirety) in the structure? I suspect that neither the ligand nor substrate are completely occupied, so should I modify the occupancies to reflect this? Thanks, Matt
