Hi Matt,
I would do this:
- obtain as good as possible refined model (to assert the noise level is
low) (I presume you refine anisotropic ADPs, include H atoms, constantly
update ordered solvent, etc...);
- remove the ligand (all conformations);
- compute mFo-DFc and 2mFo-DFc average kick maps;
- model whatever you can into these maps;
- do some refinement : coordinates+ADPs (at 1.49A I presume you do
anisotropic ADP refinement+constrained group occupancy);
- see what mFo-DFc and 2mFo-DFc maps show. You may want to compute map
CC, and values of 2mFo-DFc and mFo-DFc maps at atomic positions for all
ligand conformations.
Please let me know if you have questions about how to do the above steps
in PHENIX, or if you want to discuss more options.
Cheers,
Pavel.
On 9/23/09 4:09 PM, Matt Merski wrote:
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]
