Extended Data Fig. 8. Cell-based GCaMP assay parameters.

a,b, Relative fluorescence changes in HEK293 cells transfected with GCaMP plus wild-type (WT) or mutant Orco alone (a; titrated with VUAA1) or in the presence of An. gambiae OR65 (b; titrated with eugenol). Plots using the same symbols were collected on the same day. c,d, Fitted Hill equation parameters for Orco and Orco-OR65 (mean ± s.d). The assays were repeated N number of independent times to obtain the maximum ΔF/F_o response, concentration for half-maximal response (EC₅₀) and Hill coefficient (n). (ΔF/F_o)_norm is the fitted maximum ΔF/F_o response relative to a wild-type (WT) control Orco or Orco-OR65 experiment from the same plate (for mutations that could not be accurately fit, the measured maximum ΔF/F_o response was used). Two one-way ANOVA tests were performed using these EC₅₀ data followed by Tukey-Kramer multiple comparison tests. (1) Wild-type and mutant Orco homomers (P values are from comparisons to wild-type Orco) (2) Wild-type and mutant Orco co-transfected with OR65 (P values are from comparisons to wild-type Orco with OR65). *Fitted parameters for Orco T465A or S458A homomers + VUAA1 could not be obtained as the dose-response curves did not saturate: a lower-bound estimate for their EC₅₀ are ~3×10⁻⁴ M. **No binding was observed for Orco Y466A in the presence of OR65.