Fig. 2: Domain architecture and biochemical properties of ancestral KaiCs.

a Key residues and motifs mapped to the domain compositions of KaiC^Se and ancestral KaiCs. b Temperature dependence of the steady-state ATPase activity of ancestral KaiCs at pH 7.0 and KaiC^Se at pH 8.0. Open circles correspond to raw data shown in Supplementary Table. 1. Bar graphs show means ± s.d.; For KaiC^Se (30 °C and 40 °C), three independent purifications; For KaiC^α, KaiC^β, and KaiC^δ (30 °C and 40 °C), three replicates from a single purification; For KaiC^γ, three replicates from a single purification (30 °C) and three replicates from two independent purifications (40 °C); For KaiC^ε and KaiC^η (30 and 40 °C), six replicates from two independent purifications; For KaiC^ζ, six replicates from a single purification (30 °C) and six replicates from two independent purifications (40 °C). c Pre–steady-state analysis of the ATPase activity. Dark-colored circles correspond to the mean of separate experiments; For KaiC^α, KaiC^δ, KaiC^ε, and KaiC^ζ, three independent purifications (open squares, triangles, and diamonds); For KaiC^β, two independent purifications (open squares and triangles); For KaiC^γ, four independent purifications (open squares, triangles, inverted triangles, and diamonds). The data of KaiC^Se were taken from the previous report²⁹ and presented as mean ± s.d. of separate experiments using three independent purifications. Bioluminescent rhythm assays for Se7942 reporter strains carrying d kaiA^Se: kaiB^Se: kaiC^γ and e kaiA^Se: kaiB^Se: kaiC^β, but without kaiC^Se, were performed under continuous-light illumination condition at 30 °C. Thin and thick lines represent individual (n = 12 and 7 for (d) and (e), respectively) and average traces, respectively. The period length (32.9 ± 0.7 h) of the strain with kaiA^Se: kaiB^Se: kaiC^γ was consistent with that of the in vitro A^γB^γC^γ set at pH 7.6–7.8 at 30 °C in (f). f pH-dependence of the period length for the in vitro A^γB^γC^γ, A^δB^δC^δ, and A^SeB^SeC^Se sets at 30 °C and 40 °C. The data for the A^SeB^SeC^γ set at 30 °C at pH 7.0 (three replicates from a single purification) and pH 8.0 (three independent purifications) were presented by brown circles as means ± s.d. Each plot other than the A^SeB^SeC^γ set corresponds to a single experiment using a single purification.