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- PDB-7s67: Extended conformation of daytime state KaiC -

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Basic information

Entry
Database: PDB / ID: 7s67
TitleExtended conformation of daytime state KaiC
ComponentsCircadian clock protein kinase KaiC
KeywordsCIRCADIAN CLOCK PROTEIN / AAA ATPase / Circadian Oscillator / Kinase / Phosphatase
Function / homology
Function and homology information


regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / protein serine/threonine/tyrosine kinase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / circadian rhythm / non-specific serine/threonine protein kinase / phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity ...regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / protein serine/threonine/tyrosine kinase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / circadian rhythm / non-specific serine/threonine protein kinase / phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / regulation of DNA-templated transcription / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding
Similarity search - Function
Circadian clock KaiC, bacteria / : / Circadian clock protein kinase KaiC / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Circadian clock oscillator protein KaiC
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSandate, C.R. / Swan, J.A. / Partch, C.L. / Lander, G.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)R21GM142196 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Coupling of distant ATPase domains in the circadian clock protein KaiC.
Authors: Jeffrey A Swan / Colby R Sandate / Archana G Chavan / Alfred M Freeberg / Diana Etwaru / Dustin C Ernst / Joseph G Palacios / Susan S Golden / Andy LiWang / Gabriel C Lander / Carrie L Partch /
Abstract: The AAA family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains ...The AAA family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.
History
DepositionSep 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Aug 3, 2022Group: 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 1.3Aug 24, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Circadian clock protein kinase KaiC
B: Circadian clock protein kinase KaiC
C: Circadian clock protein kinase KaiC
D: Circadian clock protein kinase KaiC
E: Circadian clock protein kinase KaiC
F: Circadian clock protein kinase KaiC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)354,40730
Polymers348,5096
Non-polymers5,89824
Water0
1


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

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Components

#1: Protein
Circadian clock protein kinase KaiC /


Mass: 58084.781 Da / Num. of mol.: 6 / Mutation: S431A T432E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: kaiC, Synpcc7942_1216, see0011
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q79PF4, non-specific serine/threonine protein kinase
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Compressed state hexamer of nighttime state phosphomutant KaiC (KaiC-EA)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.350 MDa / Experimental value: NO
Source (natural)Organism: Synechococcus elongatus (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Plasmid: pET-28b
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium chlorideNaClSodium chloride1
31 mMAdenosine triphosphateC10H16N5O13P31
41 mMMagnesium chlorideMgCl21
51 mMTCEPC9H15O6P1
64 mMFluorinated fos-choline-8C13H17F13NO4P1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Graphene-coated grids were made hydrophilic via treatment by UV/ozone cleaner.
Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K
Details: Grids with applied sample were manually blotted with filter paper (Whatman No.1) for 3 seconds in a 4 C cold room before plunge freezing in liquid ethane cooled by liquid nitrogen.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Calibrated magnification: 43478 X / Nominal defocus max: -2000 nm / Nominal defocus min: -500 nm / Calibrated defocus min: -500 nm / Calibrated defocus max: -2000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 6.2 sec. / Electron dose: 40.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1541
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 62 / Used frames/image: 1-62

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
3Leginonimage acquisition
5RELION3CTF correction
8UCSF Chimeramodel fitting
12RELION3initial Euler assignment
14RELION3final Euler assignment
16RELION3classification
18RELION33D reconstruction
20Cootmodel refinement
CTF correctionDetails: CTF Refinement performed in Relion / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 427620
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89892 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 3K0C

3k0c

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