Cryo-EM Structure of μOR-Gi Complex

The Cryo-EM structure of the μOR–G_i complex with scFv16⁴ (PDB: 6ddf) was used as an initial model for model rebuilding and refinement. A polyalanine model was made from the μOR structure, and together with structures of G_i and sFv16 (PDB: 6ddf) was docked into the EM electron density map in Chimera⁶¹ . The resulting model was subjected to autobuilding in Buccaneer⁶² , iterative building in Coot⁶³ and refinement in Phenix.real_space_refine⁶⁴ . Initial coordinates and refinement parameters for the ligand bromocriptine were prepared with Grade web server (http://grade.globalphasing.org). The structure of the AH domain from the GDP-bound G_i crystal structure^{36 (link)} (PDB: 1GP2) was docked into the focused refined EM map. Manual editing in Coot and refinement in Phenix.real_space_refine were carried out to reduce the clashes of the docked AH domain with the Gα_i Ras-like domain and Gβ subunit. The density of T4 lysozyme was poor and not modelled. FSC curves were calculated based on the model and maps in Phenix.validatiion_cryoem (Extended Data Fig. 6c). Representative cryo-EM density for the receptor is displayed in Extended Data Fig. 6d. MolProbity⁶⁵ was used to evaluate the final structures. In the Ramachandran plot, 97.7% and 2.3% of residues were in favored and allowed regions, respectively. The statistics for data collection and refinement are included in Extended Data Table 1. The consensus cryo-EM density map and two focused maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-21243, EMD-21244, and EMD-21245. Atomic coordinates have been deposited in the PDB under accession code 6vms.