a, Size-exclusion chromatography (SEC) of MthK reconstituted in nanodiscs composed of 3:1 POPE:POPG lipids with MSP1E3. The main (highest amplitude) peak from SEC was separated using SDS–PAGE (inset), showing the components of MthK nanodiscs (indicated). b, Representative micrograph of the MthK nanodisc sample in 0 Ca2+ and 5 mM EDTA (left). Selected 2D-class averages (right). c, Final cryo-EM map of closed MthK coloured by local resolution. d, FSC curves between the two independently refined half-maps after masking (black curve) and from cross validation between the atomic model, refined against half-map 1, against masked sum of both half-maps (red). e, Angular distribution of particles used in the reconstitution. f, Density of MthK EDTA closed state. Segmented cryo-EM density maps (grey mesh) of closed MthK in the absence of Ca2+. The fitted corresponding atomic model is in cyan.