Matt, Modeling two molecules that occupy overlapping binding sites in a structure simply involves designating them as alternate conformers, with the same chain and residue number, and an occupancy that sums to 1.0. For example, if you have an AMP and an ADP that occupy the same binding site, you would define them as
AAMP B 501 BADP B 501 and initially set the occupancies for the atoms in each conformer to the ratio (50:50, 30:70, etc.) that you observe in the density. Refinement in this manner is straightforward in PHENIX. Diana ************************************************** Diana R. Tomchick Professor Departments of Biophysics and Biochemistry University of Texas Southwestern Medical Center 5323 Harry Hines Blvd. Rm. ND10.214A Dallas, TX 75390-8816 diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu> (214) 645-6383 (phone) (214) 645-6353 (fax) On Dec 13, 2017, at 1:11 PM, Matthew Bratkowski <mab...@cornell.edu<mailto:mab...@cornell.edu>> wrote: 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 ________________________________ UT Southwestern Medical Center The future of medicine, today.
