Extended Data Fig. 3: Purification of the mTAAR9–Gs–scFv16 complex and the mTAAR9–Golf–scFv16 complex.

a-d, Dose response curves of SPE (a), DMCHA (b), PEA (c), and CAD (d) induced Gαs-Gβγ dissociation in mTAAR9 or BRIL-mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). To increase receptor expression, thermostabilized cytochrome b562RIL (BRIL) was incorporated at the N-terminus of full-length mTAAR9, and the chimeric protein was found to exhibit G protein coupling activity similar to that of wild-type mTAAR9. e-h, Dose response curves of SPE (e), DMCHA (f), PEA (g), and CAD (h) induced Gαs-Gβγ or modified Gαs (modGαs)-Gβγ dissociation in mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). A modified Gαs chimera (modGαs) was generated on the basis of the mini-Gs scaffold with replacement of the N terminus of Gs (residue 1 to residue 25) with Gαi1 (residue 1 to residue 18) to facilitate the binding of scFv16 and replacing the GGSGGSGG linker at the position of original Gαs α-helical ___domain (AHD, V65-L203) with that of human Gi1 (G60-K180). In HEK293 cells, the modGαs chimera has similar pharmacological profile in G protein dissociation assay downstream of mTAAR9 activation in response to stimulations of SPE, DMCHA, PEA, or CAD. i-l, Dose response curves of SPE (i), DMCHA (j), PEA (k), and CAD (l) induced Gαolf-Gβγ or modified Gαolf (modGαolf)-Gβγ dissociation in mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). A modified Gαolf chimera was generated on the basis of the mini-Golf scaffold with replacement of the N terminus of Golf (residue 1 to residue 27) with Gαi1 (residue 1 to residue 18) to facilitate the binding of scFv16. The modGαolf was expressed and purified in insect cells to stabilize the mTAAR9 complexes for cryo-EM study. In HEK293 cells, the modified Gαolf chimera has similar pharmacological profile in G protein dissociation assay downstream of mTAAR9 activation in response to SPE, DMCHA, PEA, or CAD. m-q, Representative elution profiles of in vitro reconstituted SPE–mTAAR9–Gs–scFv16 complex (m), PEA–mTAAR9–Gs–scFv16 complex (n), PEA–mTAAR9–Golf–scFv16 complex (o), DMCHA–mTAAR9–Gs–scFv16 complex (p), and CAD–mTAAR9–Gs–scFv16 complex (q). on Superose 6 Increase 10/300 column and representative SDS-PAGE results of the size-exclusion chromatography peaks.