Purification of TRPC4_DR and negative stain EM of TRPC4_DR in amphipols.

(a) SDS-PAGE of the peak fraction of TRPC4_DR in amphipols after size exclusion chromatography. Lane 1: molecular weight marker, lane 2: protein. (b) Size exclusion chromatography profile of TRPC4_DR in amphipols. Peak 1, 2 and 3 correspond to the void volume, tetrameric TRPC4_DR and cleaved GFP, respectively. (c) Representative negative stain electron micrograph of TRPC4_DR. Scale bar, 50 nm, (d) Representative 2-D class averages. Scale bar, 10 nm. (e) 3-D reconstruction of negatively stained TRPC4_DR shown in different orientations.

Cryo-EM structure of TRPC4_DR.

(a-b) Representative digital micrograph area (a) and selected 2-D class averages (b) of TRPC4_DR embedded in vitrified ice. Scale bars, 50 nm (a), and 10 nm (b). (c) The ab initio 3-D reconstruction obtained with RVIPER. (d) Angular distribution of the particles. (e) The cryo-EM density map of TRPC4_DR colored according to the local resolution. (f) Fourier Shell Correlation (FSC) curve between maps from two independently refined half data sets (black). The 0.143 criterion indicates an average resolution of 3.6 Å. The grey curve shows the FSC curve between the final map versus the atomic model. (g) Representative regions of the density with fitted atomic model.