Random spherically constrained single-particle (RSC) method to study voltage-gated ion channels

Abstract

To study membrane protein structures using cryo-Electron Microscopy (cryo-EM), membrane proteins are usually extracted from cell membranes and solubilized in detergents. To restore the lipid bilayer environment of membrane proteins, a method called “random spherically constrained” (RSC) single-particle cryo-EM has been developed. The RSC platform establishes the lipid environment for membrane proteins and makes it possible, for the first time, to apply the desired transmembrane potential to trap voltage-gated ion channels in the desired functional states (e.g., deactivated voltage sensor at −120, mV) for structural analysis. No rupture or leakage was observed during the establishment of the transmembrane potential. The spherical geometry of liposomes is used as a constraint to accurately determine the orientation of the inserted membrane protein.