Fig. 4: Crystal structures of ancestral KaiCs.

a Similarity of the overall structures of KaiC^γ (pink), KaiC^δ (blue), and KaiC^Se (gray, PDB ID: 7DXQ). Black thick arrows indicate the viewing directions for each panel. b Superimposition of KaiC^γ (pink) and KaiC^Se (gray) using one of the CII domains (magenta star). The main-chain root-mean-squared deviation (RMSD) values of the fitted and other parts were 0.7 and 1.2 Å, respectively. For clarity, only the two most distantly located subunits within the hexamer are shown. c Superimposition of KaiC^γ (pink) and KaiC^β (yellow) using one of the CII domains (magenta star). Main-chain RMSD values of the fitted and other parts were 0.7 and 5.3 Å, respectively. d Zoomed-in views of the pre-hydrolysis CI–CI interfaces (left) for KaiC^γ (pink/orange), KaiC^δ (cyan/blue), and KaiC^Se (white/gray), and the post-hydrolysis CI–CI interface (right) for KaiC^β (yellow/brown). Dotted magenta lines represent hydrogen bonds. e Zoomed-in views of the nucleotide enclosure loop for KaiC^γ (top) and KaiC^β (bottom). In bottom panel, KaiC^γ was superimposed on KaiC^β using one CI protomer, and then only the AMP–PNP molecule (green stick model) bound to KaiC^γ is shown for comparison with the ADP molecule (magenta stick model) of KaiC^β. f Exchange with AMP–PNP was barely detected in KaiC^β-bound ADP, in contrast to KaiC^Se- and KaiC^γ-bound ADP. Ancestral KaiCs and KaiC^Se were incubated at pH 7.0 and 8.0, respectively, at 30 °C in the presence of 1 mM AMP–PNP; the fractions of nucleotides bound by KaiCs were quantified. Bar graphs show mean ± s.d. of three replicates from a single purification. g CII domains of KaiC^γ (left) and KaiC^β (right) viewed from the inner-diameter side of the hexamers. A-loop and PSw correspond to the C-terminal tail and the phosphor-switch, respectively, in KaiC. The magenta meshes represent the F_obs–F_calc omit maps contoured at 3σ. Dotted red lines represent potentially flexible parts that could not be determined because of poor electron density.