Cryo-EM structure of rat TRPV1 channel

A minimal functional rat TRPV1 construct was cloned into a modified Bacmam vector (Invitrogen) containing an N-terminal fusion cassette (Kozac-MBP-Tev protease site) for purification on amylose resin. TRPV1 protein was expressed in HEK293S GnTI⁻ cells grown in suspension at 37°C. Cells were harvested 48 hours post transduction for preparation of crude membrane and subsequent protein purification, as described^{10 (link)}. Negative stain EM and cryo-EM were carried out following established protocols^{15 (link),44} . At 3.4Å resolution, the cryo-EM map was of sufficient quality for de novo atomic model building. A poly-alanine model was first built in Coot and amino acid assignment subsequently achieved based mainly on the clearly defined side chains densities of bulky residues such as Phe, Tyr, and Trp, as well as some Arg and Lys residues.