Extended Data Fig. 2: Cryo-EM data processing of VAChT^apo.

a. Flow chart for cryo-EM data processing of VAChT^apo. A total of 3,030 micrographs were motion-corrected and dose-weighted using MotionCor2 with 7 × 10 patching in RELION-3.1. A representative motion-corrected micrograph of this dataset is shown here (scale bar = 30 nm). The particles were picked using template picker, and 2D classification was performed to remove junk particles in cryoSPARC. After three rounds of heterogeneous refinements, 218,702 particles displaying transmembrane helices were classified and subjected to Bayesian polishing in RELION-3.1 to further improve the map quality. The final map was reported at 3.4-Å resolution according to the golden standard Fourier shell correlation (GSFSC) criterion. Details of data processing can be found in Method. b. The angular distribution of the final reconstruction. c. Local resolution distribution of VAChT^apo. d. Fourier Shell Correlations (FSC) of the final map of the VAChT^apo, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM map and the structural model shown in black. e. Representative overlay of cryo-EM density and transmembrane helices of VAChT^apo. The cryo-EM maps are shown as gray surface.