Abstract
A natural way to preserve the native conformation of membrane proteins for structural study is to reconstitute them into liposomes (lipid vesicles). Here we applied this method to single-particle cryo-EM imaging of the hSlo large-conductance voltage- and Ca2+-activated potassium channel (BK channel). In an assay using the JC-1 voltage-sensitive dye, proteoliposomes develop a membrane potential that is equal to that produced by valinomycin, and to that of empty liposomes when the BK channels are blocked by barium (A). For cryo-EM imaging the reconstituted BK proteoliposomes were tethered to a 2D streptavidin crystal (B). From this image the periodic crystal information was removed (C) and a liposome model of electron scattering was used to subtract the lipid bilayers (D). For 3D reconstruction, single-particle images (boxes) were oriented with constraints based on the spherical vesicle geometry. A structure based on these particle images will be presented. Scale bar in the figures is 50 nm.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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