Structures of the cyanobacterial
circadian oscillator frozen in a fully
Joost Snijder,1*† Jan M. Schuller,2*‡ Anika Wiegard,3 Philip Lössl,1
Nicolas Schmelling,3 Ilka M. Axmann,3 Jürgen M. Plitzko,2
Friedrich Förster,2,4§ Albert J. R. Heck1§
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 5ʹ-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.
Many organisms, from cyanobacteria to animals, have adapted to Earth’s day- night cycle with the evolution of an en- dogenous biological clock. These clocks enable circadian rhythms of gene expression and metabolism with a period close to
24 hours. Many circadian rhythms rely on complex networks of transcription-translation feedback, but simpler posttranslational oscillations
have also been described in both cyanobacteria
and human red blood cells (1). The circadian
oscillator of cyanobacteria is composed of three
components: the proteins KaiC, KaiB, and KaiA
(2). This posttranslational oscillator is robust enough
to allow reconstitution simply through incubation
of purified recombinant KaiC, KaiB, and KaiA in
the presence of adenosine 5′- triphosphate (ATP) (3).
The in vitro oscillator can maintain a stable rhythm
for weeks (4, 5), allowing for its detailed study.
The proteins of the Kai system collectively gen-
erate a circadian rhythm based on assembly dy-
namics associated with KaiC autophosphorylation
and dephosphorylation (6, 7). KaiC forms a homo-
hexamer consisting of two stacked rings of domains
CI and CII, which have adenosine 5′ - triphosphatase
(ATPase) and kinase activity, respectively (8). Dur-
ing the subjective day, the kinase activity of KaiC is
stimulated by the binding of KaiA to the intrin-
sically disordered C-terminal regions of KaiC,
resulting in sequential autophosphorylation at
Thr432 and Ser431 of KaiC (9). During the subjec-
tive night, KaiB interacts with phosphorylated
KaiC, forming the KaiCB complex (8). Binding of
KaiB to KaiC changes the activities of SasA and
CikA, which are key signaling proteins of clock-
output pathways that modulate transcription (10).
Moreover, the KaiCB complex exposes an addi-
tional KaiA-binding site, sequestering KaiA and
thus preventing its productive association with
KaiC (11). The sequestration of KaiA allows KaiC
to readopt its default autodephosphorylation ac-
tivity, thereby slowly resetting the protein clock to
an unphosphorylated state (4).
Atomic-level structures of the individual Kai proteins are available (12–14), but structural information on the KaiCB and KaiCBA complexes is
still ambiguous (15–18). KaiB forms monomers,
dimers, and tetramers in solution, with six KaiB
monomers binding cooperatively to one KaiC hexamer (19). It has been unclear whether KaiB binds
to the CI or CII domain of KaiC (11, 15, 19–21).
Nuclear magnetic resonance (NMR) spectroscopy
studies of engineered and truncated Kai proteins
suggested that KaiB binds the KaiC-CI domain
and only one subunit of a KaiA dimer (11), but it
is unclear whether the wild-type, full-length proteins arrange similarly in the KaiCBA complex.
Here we use mass spectrometry (MS) and cryo–
electron microscopy (cryo-EM) to study the as-
sembly and structures of the full-length clock com-
ponents to provide a structural basis for the assembly
dynamics of the in vitro circadian oscillator.
The standard in vitro Kai oscillator consists of
a 2:2:1 molar ratio of KaiC:KaiB:KaiA in the
presence of excess MgATP, incubated at 30°C (3).
We tracked the phosphorylation-dependent as-
sembly of the Kai proteins under these condi-
tions and used native MS to determine the masses
and stoichiometries of the formed noncovalent
assemblies (22). For the in vitro Kai oscillator, we
simultaneously detected multiple co-occurring
Kai-protein complexes, revealing more than 10
different Kai protein–assembly stoichiometries
over the course of 24 hours (Fig. 1A and table S1).
The KaiC starting material had low amounts
of phosphorylation. Upon initial mixing, most KaiC
therefore existed as a free hexamer, whereas a
small fraction formed a complex with one or two
KaiA dimers (fig. S1A). These KaiCA complexes
have autophosphorylation activity, which led to
cooperative formation of phosphorylated KaiC6B6
complexes through a KaiC6B1 intermediate (19).
In our samples, formation of KaiCA and KaiCB
complexes peaked at 4 to 8 hours incubation time.
The formation of higher-order KaiCBA complexes
followed the formation of KaiCB complexes, with
a maximum at 8 to 12 hours of incubation fol-
lowed by a steady decline toward 24 hours. We
observed KaiC6B6 with between one and six KaiA
dimers bound. Detailed assignments of peaks and
repeated measurements are shown in Fig. 1B and
figs. S1 to S3. During the dephosphorylation phase
(16 to 24 hours), as KaiCBA complexes disassem-
ble, we detected KaiA2B1 complexes in the lower-
mass region of the spectra (fig. S1B). Thus, the
disassembly pathway of the KaiCBA complexes
appears not to be simply the reverse of the as-
sembly pathway but rather a distinct route.
We attempted to freeze Kai-protein assembly
in specific states, producing particles amenable
to more detailed structural characterization.
Whereas at 30°C the default activity of KaiC is
autodephosphorylation, autophosphorylation is
favored at 4°C (7, 17). Therefore, we tested how a
lower incubation temperature affected assembly
of the complete in vitro oscillator. At 4°C, KaiCBA-complex formation was slower than at 30°C.
However, KaiCBA abundance steadily increased,
and, even after 24 hours, it did not peak (Fig. 1A).
This indicated a possible route for preparation
of KaiCBA complexes with full occupancy of the
KaiA-binding site. Therefore, we incubated KaiC,
KaiB, and KaiA at a 1:3:3 molar ratio at 4°C for
one week in the presence of MgATP. We observed
near-complete occupancy of the KaiA-binding
site, as seen from the predominant formation of
KaiC6B6A12 assemblies (Fig. 1C). The measured
mass of this complex was 823.3 ± 0.5 (standard
deviation) kDa, compared to a theoretical mass
of 821.3 kDa for KaiC6B6A12 (table S1). Similarly,
prolonged incubation of KaiC with KaiB at 4°C
resulted in the efficient formation of KaiC6B6
complexes (measured: 426.9 ± 0.1 kDa; theoretical:
426.4 kDa; table S1). Further experiments revealed
that formation of the KaiCB complex is the limiting
step for the complete assembly of KaiCBA (fig. S1C).
1Biomolecular Mass Spectrometry and Proteomics and
Netherlands Proteomics Center, Bijvoet Center for
Biomolecular Research, and Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, Padualaan 8,
3584 CH, Utrecht, Netherlands. 2Max Planck Institute of
Biochemistry, Department of Molecular Structural Biology,
D-82152 Martinsried, Germany. 3Institute for Synthetic
Microbiology, Cluster of Excellence on Plant Sciences
(CEPLAS), Heinrich Heine University Düsseldorf, D-40225
Düsseldorf, Germany. 4Cryo-electron Microscopy, Bijvoet
Center for Biomolecular Research, Utrecht University,
Padualaan 8, 3584 CH, Utrecht, Netherlands.
*These authors contributed equally to this work. †Present address:
Department of Biochemistry, University of Washington, Seattle,
WA 98195, USA. ‡Present address: Max Planck Institute of
Biochemistry, Department of Structural Cell Biology, D-82152
§Corresponding author. Email: a. firstname.lastname@example.org (A.J.R.H.);