Structural and Functional Analysis of M3 Muscarinic Receptor

The M₃ muscarinic receptor-T4 lysozyme fusion protein was expressed in Sf9 insect cells and purified by nickel affinity chromatography followed by FLAG antibody affinity chromatography and then size exclusion chromatography. It was crystallized using the lipidic cubic phase technique, and diffraction data were collected at the GM/CA-CAT beamline at the Advanced Photon Source at Argonne National Lab. The structure was solved by molecular replacement using merged data from 76 crystals. All-atom classical molecular dynamics simulations with explicitly represented lipids and water were performed using the CHARMM force field^{29 (link)} on Anton³⁰ . Ligand-binding simulations included no artificial forces. Dissociation studies included a time-varying biasing term that gradually forces the ligand away from its crystallographic position, but not along any prespecified pathway or direction. Full details are provided in the online methods.