Extended Data Fig. 2: Dynamic residency of Gα AHD in open and closed positions.

a, Measurement of the real time of vitrification using a Vitrobot. The Vitrobot timing is the sum of user programmed blot time and wait time, 2 sec (4.95 sec ± 0.026 S.E.M., n = 10), 7 sec (9.99 sec ± 0.029 S.E.M., n = 10), 14 sec (17.02 sec ± 0.040 S.E.M., n = 10), where n indicates number of measurements recorded. Individual data points shown. b-h, To determine the residency of the AHD between open and closed positions in cryo-EM reconstructions, the AHD was docked into frames 1 (maximally open AHD) and 20 (maximally closed AHD) of each 3DVA trajectory (c-d, f-h) or 3D classes ordered from left, class A, to right, class T, by percent contribution of particles from the 17 sec dataset (e), a region of 6 Å from the docked structures was used to define ‘fully open’ or ‘fully closed’ respectively, b, and the volume of cryo-EM map at a threshold level of 0.05 that was enclosed in the defined regions was determined, c-g. i, Location of Gα AHD in relation to Gβ. The crystal structure (PDB:3SN6) locates the Gα AHD (grey) adjacent to Gβ blades 1 (red) and 2 (orange) and interacting with blade 2. In contrast, the location of the cryo-EM density that corresponds to the AHD lies adjacent to Gβ blades 2 and 3 (yellow) in both the nucleotide-free and GTP conditions. The cryo-EM structure of NTSR1-Gi also has an open AHD adjacent to blades 2 and 3, but in a different orientation. Structures have been aligned to Gβ. In the middle panels, the cryo-EM density envelope (Gaussian filtered, σ = 2) of the unsharpened map is shown with the density corresponding to the location of the AHD shaded in grey.