Cyanobacterial clock proteins; KaiA, KaiB and KaiC are periodically assembled into the large complex (ABC complex) in a 24-hour period in vitro. A cryo-EM study reported the structure of ABC complex. However, the locations of N-terminal domains of KaiA (AN domains) have not been resolved due to their high flexibility. For a better understanding of the assembly mechanisms, we investigated the overall structural model by integrating structural modelling based on the cryo-EM structure and small-angle X-ray/neutron scattering (SAXS/SANS) measurements. From the profiles of size exclusion chromatography (SEC)-SAXS measurements, the structural candidates of ABC complex were classified into three types (Type 1 ~ Type 3) from the viewpoint of the coordinate structure of AN domains. In order to refine the classified structures by the spatial arrangement of AN domains, we applied the SEC-inverse contrast matching (iCM)-SANS measurement to selectively observe KaiA in ABC complex which was prepared with hydrogenated KaiA along with 75%-deuterated KaiB and KaiC. The scattering from the latter components diminished and only the former component was visible in 100% D2O. By comparing the calculated SEC-iCM-SANS profiles for classified models and the experimental data, Type I were clearly the best structural model. In this report, we show the advantage of SEC-iCM-SANS measurement to resolve a large complex harboring dynamically fluctuating domains.
