Hi, Part of the problem is that you are likely dealing with not just two discrete conformations of the ligand (phenylpropanol by the looks of it) and protein - there may be 'transition' states occupied below the level of detection - therefore between the two 'end' states there's probably a smeared continuum, and the sum of the two discretely modeled occupancies is not 1 (integral over the smear would be 1 of course but you're not modeling this). To make things worse, the floppy propanol chain is probably sampling additional sub-conformations of its own...
Notably, since the ligand is anchored via its phenyl ring, and the transition between two states does not perturb the apparent occupancy of the ring atoms (even though in strict sense there are multiple sets of atoms superposed with each set having unique vibraitonal modes and so forth). Q1: for the purposes of whatever research you're doing I would argue that modeling the minor ligand position is acceptable. Q2: local omit map, kick-omit map, and the density-filling procedure using partially occupied random atoms followed by refinement (CCP4 post from about 7-8 months ago mentioned that very nice paper which I have since forgotten, alas) - these methods may give you maps with some more clarity. Good luck, Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Matt Merski Sent: Wednesday, September 23, 2009 6:10 PM To: [email protected] Subject: [ccp4bb] Modeling Multiple Ligand Conformations Hi all, I am solving a series of protein-ligand complex structures in which the larger ligands typically cause an expansion of the binding site, changing the receptor into an open conformation, while the smaller ligands do not change receptor conformation upon binding. In one structure (1.49 Ang resolution), after several iterations of refinement with phenix (R-work = 0.1609, R-free = 0.1918), I see both conformations: all of the closed receptor conformation and the backbone conformation and 6 out of 7 of the side chains of the open conformation can be seen in the 2Fo-Fc map (70% occupancy for the closed conformation, 30% for the open conformation). Modeling in the ligand as one 100% occupancy pose that fits in the closed receptor conformation (Fig. 1) gives some negative Fo-Fc density up to sigma = 3.8 and does not explain the appearance of the open receptor conformation. If I model the ligand in at 70% occupancy, the negative 2Fo-Fc density is eliminated and some positive Fo-Fc density appears around the ligand (Fig.2), suggesting the presence of a second ligand conformation but there is not enough density to unambiguously place a second ligand conformation (corresponding to the open receptor conformation). When I model in a second ligand conformation (Fig. 3), then after refinement positive Fo-Fc density disappears without any negative Fo-Fc density appearing but no 2Fo-Fc density appears after refinement to confirm the correctness of the ligand pose. Q #1: should I model the ligand in if it eliminates the Fo-Fc positive density and doesn't cause negative density? Or should I leave it out despite the indirect evidence from the altered receptor conformation for this additional pose? Because this is a series of ligands we know that the larger receptor conformation implies that a ligand is present in a pose that opens up the binding site but it's difficult to confirm the second ligand pose. Q #2: Is there any way to directly test the correctness of the second pose that is not dependant on the 2Fo-Fc maps? Is there some kind of statistical test (such as a local R-factor) to show that the pose is not in direct disagreement with the data? A pdf of the figures is available at http://blur.compbio.ucsf.edu/~merski/figs.pdf Thanks for your help. Matthew Merski UCSF Dept. of Pharmaceutical Chemistry [email protected]
