Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.
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