ATPase enzymatic assays were used to determine changes in ADP concentration over time. At time zero, 10 mM ATP (containing 50 μM ADP) and 10 mM MgCl2 were added to the reaction mixture that contained purified cysteine free E. coli F1F0 ATP synthase and incubated at 22°C. At 15, 30, 45, 60, 120 and 240 s post-ATP addition aliquots of the assay mixture were extracted, and ATP and ADP were quantified using HPLC-UV, with 50 μM ADP subtracted as due to baseline contamination (see Materials and methods). The assay was performed in biological triplicate, using three different protein purifications (labelled black, red and blue). After 45 s, at which point samples were frozen for this cryo-EM study, ~0.25 mM ATP was hydrolysed, and the rate of ADP production was linear. The total concentration of ADP at 45 s was ~0.3 mM.