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
> [log in to unmask]
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