Saturation-binding reactions were incubated at 37°C for 24 h, whereas binding-competition reactions were incubated at 37°C for 24 or 48 h. All individual curves for a given combination of construct and ligand were globally fitted (regardless of incubation duration), and parameter standard errors were obtained from these fits (Fig. S4). For competition experiments, the ratio between the fixed and half-saturation concentrations of [¹²⁵I]-α-BgTx was approximately unity. The number of independent saturation or competition assays contributing to each parameter estimation is indicated in parentheses. Parameter estimates obtained from incubations at 4 or 22°C are not listed.