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- PDB-8fwj: Structure of dodecameric KaiC-RS-S413E/S414E complexed with KaiB-... -

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Basic information

Entry
Database: PDB / ID: 8fwj
TitleStructure of dodecameric KaiC-RS-S413E/S414E complexed with KaiB-RS solved by cryo-EM
Components(Circadian clock protein ...) x 2
KeywordsCIRCADIAN CLOCK PROTEIN / autokinase
Function / homology
Function and homology information


rhythmic process / kinase activity / non-specific serine/threonine protein kinase / phosphorylation / ATP hydrolysis activity / ATP binding
Similarity search - Function
Circadian clock protein KaiB-like / KaiB domain / KaiB domain / KaiB / Circadian clock protein kinase KaiC / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC ...Circadian clock protein KaiB-like / KaiB domain / KaiB domain / KaiB / Circadian clock protein kinase KaiC / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / Thioredoxin-like superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Circadian clock protein KaiB / non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesCereibacter sphaeroides (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsPadua, R.A.P. / Grant, T. / Pitsawong, W. / Hoemberger, M.S. / Otten, R. / Bradshaw, N. / Grigorieff, N. / Kern, D.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2023
Title: From primordial clocks to circadian oscillators.
Authors: Warintra Pitsawong / Ricardo A P Pádua / Timothy Grant / Marc Hoemberger / Renee Otten / Niels Bradshaw / Nikolaus Grigorieff / Dorothee Kern /
Abstract: Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary ...Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.
History
DepositionJan 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 19, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Circadian clock protein KaiC
B: Circadian clock protein KaiC
C: Circadian clock protein KaiC
D: Circadian clock protein KaiC
E: Circadian clock protein KaiC
F: Circadian clock protein KaiC
G: Circadian clock protein KaiC
H: Circadian clock protein KaiC
I: Circadian clock protein KaiC
J: Circadian clock protein KaiC
K: Circadian clock protein KaiC
L: Circadian clock protein KaiC
M: Circadian clock protein KaiB
N: Circadian clock protein KaiB
O: Circadian clock protein KaiB
P: Circadian clock protein KaiB
Q: Circadian clock protein KaiB
R: Circadian clock protein KaiB
S: Circadian clock protein KaiB
T: Circadian clock protein KaiB
U: Circadian clock protein KaiB
V: Circadian clock protein KaiB
W: Circadian clock protein KaiB
X: Circadian clock protein KaiB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)887,59572
Polymers875,79924
Non-polymers11,79648
Water86548
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Circadian clock protein ... , 2 types, 24 molecules ABCDEFGHIJKLMNOPQRSTUVWX

#1: Protein
Circadian clock protein KaiC /


Mass: 62666.133 Da / Num. of mol.: 12 / Mutation: S413E, S414E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cereibacter sphaeroides (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B9KWX8
#2: Protein
Circadian clock protein KaiB /


Mass: 10317.127 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cereibacter sphaeroides (bacteria) / Gene: EBL86_22425 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A3G6WWB7

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Non-polymers , 4 types, 96 molecules

#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 48 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.875 MDa / Experimental value: NO
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 6.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 100 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k)

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.5/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Linux / Type: package
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4cisTEM2.0.0-alphaCTF correction
9cisTEM2.0.0-alphainitial Euler assignment
10cisTEM2.0.0-alphafinal Euler assignment
11cisTEM2.0.0-alphaclassification
12cisTEM2.0.0-alpha3D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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