Extended Data Fig. 4: Cryo-EM data processing of VAChTVES.

a. Flow chart for cryo-EM data processing. A total of 1,762 movie stacks were collected and motion-corrected. A representative motion-corrected micrograph of this dataset is shown here (scale bar = 30 nm). A total of 2,257,399 particles were picked using template picker in CryoSPARC, and were used for 2D classification. 2D class averages of distinct secondary structure features from different views of VAChTVES are shown. Only classes featuring transmembrane helices were selected and subjected to heterogeneous refinements to remove junk particles, followed by local refinement to improve map quality. The final map was reported at 2.7 Å according to the GSFSC criterion. Details of data processing can be found in Method. b. The angular distribution of the final reconstruction. c. Local resolution distribution of VAChTVES. d. Fourier Shell Correlations (FSC) of the final map of the VAChTVES, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density and the structural model shown in black. e. Representative overlay of cryo-EM density and transmembrane helices of VAChTVES. The cryo-EM maps are shown as grey surface.