Abstract<br />
The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC‐binding EnvZ‐like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer) and occasionally as a hexamer (SasA6mer) in vitro. Previously, the molecular mass of the SasA‐KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel‐filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer‐KaiC hexamer (KaiC6mer) complex with a 1:1 molecular ratio. To clarify the structure of the SasA‐KaiC complex, we analyzed KaiCDD with 0.1 mmol/L ATP and 5 mmol/L MgCl2 (Mg‐ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg‐ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer) and KaiCDD6mer, respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer, respectively. The AFM images of the SasA‐KaiCDD mixture indicated no