Extended Data Fig. 1: Reconstitution of ADGRG2-Gs complex.
From: Structures of the ADGRG2–Gs complex in apo and ligand-bound forms
a, SDS-PAGE of samples of in vitro reconstituted apo-ADGRG2-AA-120-CT in complex with wild-type Gs heterotrimer, dominant negative Gs (DNGs) heterotrimer, or DNGs heterotrimer stabilized with Nb35 after Flag-tagged antibody pulling down. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. b, ADGRG2-120-AA-CT (120-CT) showed higher expression yield in Sf9-insect cell expression system compared with wild-type ADGRG2. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. c, Representative concentration dependent curve of the VPM-p15-induced cAMP accumulation in HEK293 cells overexpressing similar levels (as shown in Extended Data Fig. 1d) of wild-type ADGRG2 or ADGRG2-120-AA-CT using Glosensor assay. Values are the mean ± SEM of three independent experiments performed in triplicate (n = 3). d, ELISA experiments showing similar expression levels of wild-type ADGRG2 and ADGRG2-120-AA-CT on HEK293 cell surface when indicated amounts of plasmids were transiently transfected. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). Comparison between ADGRG2-WT and ADGRG2-AA-CT was determined by two-sided one-way ANOVA with Tukey’s test. ns, no statistical significance (P = 0.7149). e-f, Representative elution profile and SDS-PAGE of the size-exclusion chromatography peak of apo-ADGRG2-AA-120-CT-DNGs (apo-ADGRG2-FL-DNGs) (e) or DHEA-ADGRG2-βT-120-CT-DNGs (DHEA-ADGRG2-βT-DNGs) (f) complex. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. g, Two-dimensional class averages of negative staining sample showing distinct N-terminal structural features of the apo-ADGRG2-FL-DNGs complex. Scale bar: 10 nm. h-j, Representative cryo-EM micrograph (scale bar: 20 nm) and 2D class averages (scale bar: 50 nm) of the apo-ADGRG2-FL-DNGs (h), DHEA-ADGRG2-βT-DNGs (i), and DHEA-ADGRG2-AA-120-CT-mini-Gs (DHEA-ADGRG2-FL-mini-Gs) (j) complexes. The 2D class average for apo-ADGRG2-FL-DNGs complex was shown in Fig. 1b. Representative Cryo-EM micrographs from 9,997 movies (h) or 5,222 movies (i) or 4,780 movies (j) and representative two-dimensional class averages determined using approximately 3.46 million (i) or 0.66 million (j) particles from 2D classification were shown. k-m, Schematic flow chart of the image processing steps for the apo-ADGRG2-FL-DNGs (k), DHEA-ADGRG2-βT-DNGs (l), and DHEA-ADGRG2-FL-mini-Gs (m) complexes.
