(A) Side view of the RTx/DkTx bound cryo-EM structures of TRPV1 refined using the NMR structure of DkTx (Bae et al., 2016). The EM density tentatively assigned to RTx is colored orange and the backbone of DkTx is colored bright purple. (B) Close-up view of the RTx EM density (orange) with residues closer than 12.5 Å from the center of mass of that density shown in stick representation. The four residues studied here (S512, T550, M547, E570) are colored red and the others yellow. (C) In-gel fluorescence of SDS-solubilized membranes from S. cerevisiae cells overexpressing GFP-tagged TRPV1 domain constructs using the following abbreviations: FL (Full-lenth), p.m. (pore mutant; deletion of 629–647)(Garcia-Sanz et al., 2004), N-S4 (N terminus through 575), N-S6 (N-terminus through 704), S1-S6 (423 to 704), S1-C (423 to C terminus) and S1-S4 (431 to 575). (D) Fluorescence-coupled size exclusion chromatography (FSEC) traces of GFP-tagged TRPV1 S1-S4 domain solubilized in DDM. The first peak is the void volume and the second corresponds to monomeric domain. (E) 3H-RTx binding to intact S. cerevisiae cells expressing full-length TRPV1 and its separate domains, all of which contain GFP tags on their C-termini. Data were normalized to number of cells because the trucated constructs express to higher levels compared to TRPV1, and data were corrected for non-specific binding as described in Materials and methods. Data points are the mean + S.E.M. for triplicate determinations. Smooth function for full-length GFP-tagged TRPV1 is a fit of the Hill equation to the data with Kd and Hill slope (nH) values of 7.0 nM and 0.78 for TRPV1. (F) Binding of 3H-RTx to S. cerevisiae cells containg GFP-tagged TRPV1, N-terminus His-tagged TRPV1 (without GFP), or dual GFP- and His-tagged TRPV1 construct harboring the Y511A mutation. Smooth functions are fits of the Hill equation to the data with Kd and nH values of 7.2 nM and 0.82 for TRPV1, 30 nM and 0.6 for TRPV1-His, and >238 nM and 1.1 for TRPV1 Y511A. Data points are the mean + S.E.M. for triplicate determinations.