Extended Data Fig. 3. Cryo-EM analysis of ATP-bound mtHsp60^V72I.

(a) Representative 2D class averages from the mtHsp60^ATP dataset. Scale bar equals 100 Å. Top views of single ring complexes are indicated (*). (b) Cryo-EM processing workflow for structures obtained from the mtHsp60^ATP dataset. The mask used for focused classification is shown in transparent yellow with the consensus map. Protomers from focused classification maps are colored in green (apical domain facing upward), red (apical domain facing downward), or gray (disordered apical domain). Class 1 was selected for refinement based on visual assessment of map quality. (c) View of an apical domain from the unsharpened mtHsp60^ATP consensus map and associated model. (d) Nucleotide binding pocket of mtHsp60^ATP, showing density for ATP and the γ-phosphate thereof, and Mg²⁺ and K⁺ ions (gray, from sharpened map). (e) Overlay of consensus mtHsp60^apo and mtHsp60^ATP models, aligned by the equatorial domain, showing a downward rotation of the intermediate and apical domains in the ATP-bound state. (f) Inter-ring interface of the sharpened mtHsp60^ATP consensus map and fitted model, showing contact at the left interface mediated by helix P, but no contact at the right interface. Each protomer is colored a different shade of purple. (g) Unsharpened map and model of ordered apical domains of mtHsp60^ATP focus. ‘Down’ protomers are colored purple, ‘up’ protomers are colored pink. (h) Modeling of two adjacent ATP-bound ‘up’ (left) or ‘down’ (right) protomers, generated by aligning a copy of chain C of mtHsp60^ATP focus with chain D (up pair) or a copy of chain D with chain C (down pair). A large clash is observed with two adjacent down protomers, while two adjacent up protomers appear compatible.