Fig. 3: CRD linker acts as a brake on activation.
a, smFRET histograms across increasing concentrations of glutamate, as in Fig. 1, using the CRD twisting sensor. Data are presented as mean ± s.e.m.; four to five movies per histogram; total number of traces per histogram is shown in the figure; two additional biological replicates are shown in Extended Data Fig. 6. b, Proportion of each smFRET distribution occupying the higher of two FRET states, for either the lower-lobe reporter, shown in Fig. 1 (blue), or the CRD linker reporter (green); bimodal fit carried out on the mean FRET distribution across three biological replicates, with error bars corresponding to s.e.m. Dashed line indicates theoretical correction to the proportion of high-FRET particles for the LBD twist sensor (Methods). c, Forward-rate constants, reverse-rate constants and equilibrium constants estimated for three conditions (10 µM Glu, 10 mM Glu and 20 µM LY379268) for the LBD and CRD twisting sensors across four biological replicates (n = 4); *P < 0.05 estimated with Mann–Whitney U tests (one-sided; P = 0.015 in all cases). Number of traces analyzed per biological replicate shown in Supplementary Table 1. d, Addition of the PAM BINA (orange) on top of 10 mM Glu introduces a higher FRET peak at ~0.65, whereas addition of the inhibitory heterotrimeric G protein (Gi, blue) spreads to even higher FRET values; 0 mM glutamate (gray) and 10 mM glutamate (purple) are replotted smFRET distributions from a; data are presented as mean ± s.e.m.; ≥3 movies per histogram. e, Representative smFRET traces for each condition shown in c; idealizations (red lines) are Viterbi paths generated via HMM analysis applied to each condition using ebFRET.
