Figure 2. Cryo-electron microscopy (cryo-EM) structures of the M₄R-G_i1-scFv16 complexes.

(A) Cryo-EM maps of Ipx-bound M₄R-G_i1-scFv16 complex with views from the membrane and the extracellular surface. Cryo-EM maps of the other ligand-bound structures are shown in Figure 2—figure supplement 1. (B) Representative EM density around the ligands in this study. EM-maps of Ipx-, LY298-Ipx-, and VU154-Ipx were set to a contour level of 0.011 and the receptor-focused map of ACh- was set to 0.32. (C–E) Comparison of the receptor models with bound ligands and views from the (C) membrane, (D) extracellular surface, and (E) intracellular surface.

Figure 2—figure supplement 1. Cryo-electron microscopy (cryo-EM) structures of the M₄R-G_i1-scFv16 complexes.

(A–C) Cryo-EM maps of (A) VU154-Ipx, (B) LY298-Ipx-, and (C) ACh-bound M₄R-G_i1-scFv16 complex with views from the membrane and the extracellular surface. The comparison of receptor models is shown in Figure 2. (D) Comparison of the positions of Gα_i1Gβ₁Gγ₂-scFv16 from all four cryo-EM structures with views from the membrane and extracellular surface.

Figure 2—figure supplement 2. Cryo-electron microscopy (cryo-EM) data processing and analysis.

(A–D) Flowchart of cryo-EM data processing of the (A) Ipx-, (B) VU154-Ipx-, (C) LY298-Ipx-, and (D) ACh-bound M₄ muscarinic acetylcholine receptor (mAChR) complexes with G_i1-scFv16 including particle selections, 2D and 3D classifications, EM density map, and the Fourier shell correlation (FSC) curves.

Figure 2—figure supplement 3. Cryo-electron microscopy (cryo-EM) density maps.

(A) EM maps colored by local resolution. (B–E) Representative EM density and modeling for the 7 transmembrane (TM) helices, the C-terminus of Gα_i1, and ligands for the (B) Ipx-, (C) VU154-Ipx-, (D) LY298-Ipx-, and (E) ACh-bound M₄ muscarinic acetylcholine receptor (mAChR) complexes. EM-maps of Ipx-, LY298-Ipx-, and VU154-Ipx were set to a contour level of 0.011 and the receptor-focused map of ACh- was set to 0.32.

Figure 2—figure supplement 4. Comparison of active state muscarinic acetylcholine receptor (mAChR) structures.

(A) Comparison of the Ipx- and LY298-Ipx-bound M₄ mAChR structures to the prior structures of Ipx-bound M₁ mAChR and LY2119620-Ipx-bound M₂ mAChR cryo-EM structures. Protein Data Bank (PDB) accession codes for the M₁ mAChR (PDB: 6OIJ) and the M₂ mAChR (PDB: 6OIK). (B, C) Views from the (B) extracellular and (C) intracellular surfaces. (D) Comparison of the binding pose of LY2119620 at the M₂ mAChR and LY2033298 at the M₄ mAChR. (E) Comparison of the Ipx binding site residues.

Figure 2—figure supplement 5. Comparison of active state M₄ muscarinic acetylcholine receptor (mAChR) structures.

(A) Comparison of LY298-Ipx bound M₄ mAChR structure (PDB: 7TRP, receptor colored green, Ipx blue, and LY298 cyan) to the LY2119620-Ipx bound M₄ mAChR structure (PDB: 7V68, receptor colored pink, Ipx cyan, and LY2119620 blue) (Wang et al., 2022). (B–D) View of the allosteric binding site from the top of the receptor. (B) Comparison of key allosteric residues F186^45.51 and W435^7.35 showing different positions of the residues between M₄ mAChR structures. (C) Overlay of the EM map (EMD-26100, colored gray) onto the LY298-Ipx bound M₄ mAChR structure contoured at 0.012. (D) Overlay of the EM map (EMD-31738, colored gray) onto the LY2119620-Ipx bound M₄ mAChR structure contoured at 0.15. There is a lack of EM density surrounding the allosteric residues F186^45.51 and W435^7.35 at this level of contour and all others. (E–G) View of the orthosteric binding site from the top of the receptor. (E) Comparison of key orthosteric binding site residues. (F) Related to (C) with view from orthosteric site and the EM-map contoured at 0.010. (G) Related to (D) with view from the orthosteric site with mismodeled residues. (H–K) DAQ scores provide an estimation of the local quality of protein models from cryo-electron microscopy (cryo-EM) maps on a per residue basis. DAQ scores were determined from the DAQ web server using the recommended default settings (Terashi et al., 2022). (H, J) DAQ scores from the analysis of (H) the LY298-Ipx-M₄R-G_i1 complex and (J) the LY2119620-Ipx-M₄R-G_i1 complex mapped onto the cartoon of the receptor chain and color coded by score. A DAQ score that is positive (colored blue at values of 1) indicates a correct assignment. A DAQ score near 0 (colored white) indicates a position in the map that lacks a distinct density pattern for the assigned amino acid. DAQ scores less than 0 (colored red at –1) indicate a position that could be misassigned or poorly fit. (I) DAQ scores for all four M₄ mAChR structures reported in this article with DAQ scores of each Cα atom plotted for each residue. Key orthosteric and allosteric residues are denoted by asterisks. Nearly every residue has a value above 0. (K) Similar to (I), but for all three M₄ mAChR structures reported in Wang et al., 2022. Very few residues have a score above 0, indicating potential issues with the model and maps.