Extended Data Fig. 8: Cryo-EM structure of the ELA-APJR^F101A–Gi complex and analysis of activation motifs.

a, Representative cryo-EM image of from 9,096 micrographs the ELA-APJR^F101A-Gi complex. Scale bar, 50 nm. b, Representative 2D averages showing distinct secondary structural features from different views of the ELA-APJR^F101A-Gi complex. c, Cryo-EM data processing workflow. The data was all processed by CryoSPARC and images of density maps were created in UCSF Chimera. The final 3D density maps are colored according to the local resolution. Gold-standard FSC curves from Phenix indicate overall nominal resolutions of 3.16 Å using the FSC = 0.143 criterion. d, Elution profile of APJR^F101A (comprising residues 1-350), Gα_i, Gβ, Gγ and scFv16 after SEC purification. The collected fraction for cryo-EM sample were marked between dashed lines. The gel image of APJR^F101A-Gi complex in the presence of ELA-32 (labeled in red color) compared with APJR^WT-Gi complexes in the presence of cmpd644 and ELA-32 respectively. The three part of gel images were from different gels and split with white boarder. e, Atomic model of ELA-APJR^F101A-Gi complex and global fitting of the structure into the cryo-EM density map (scFv16 is omitted for clarity). f, Superposition of ELA-_monAPJR^F101A-Gi with ELA-_monAPJR^WT-Gi complex structures. g, Conformational rearrangements in the activation-related key motifs. PIF and ‘toggle switch’ residues in the active⁻state ProtA^cmpd644 (blue) show side⁻chain movement compared to the inactive-state APJR (pink), left. Conformational rearrangement related to the Na⁺ pocket. D75^2.50 forms polar interaction with N46^1.50 in the active-state structure (ProtA^cmpd644, blue). Dashed lines represent polar interaction, middle. Conformational changes in DRY and NPxxY motif, right. The hydrogen bond is depicted as a dashed line. Related residues are presented as sticks. h, Comparison of helix 8 (H8) in ProtB^cmpd644 from _dimAPJR^cmpd644-Gi with that in AT2R (PDB ID: 5UNF). The H8 showed inverted orientation in both structures.

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