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- PDB-7s66: Extended conformation of nighttime state KaiC -

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Basic information

Entry
Database: PDB / ID: 7s66
TitleExtended conformation of nighttime 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'-TRIPHOSPHATE / Circadian clock oscillator protein KaiC
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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 1.1Jan 26, 2022Group: Experimental preparation / Category: em_sample_support
Revision 1.2Jul 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 1.3Aug 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 1.4Aug 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)354,88730
Polymers348,5096
Non-polymers6,37824
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: S431E T432A
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.: 12 / 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
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: Extended 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 solution
IDSpecimen-IDpH
117.4
227.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium chlorideNaClSodium chloride1
31 mMAdenosine triphosphateC10H16N5O13P31
41 mMMagnesium chlorideMgCl21
51 mMTCEPC9H15O6P1
620 mMTrisC4H11NO32
7150 mMSodium chlorideNaClSodium chloride2
81 mMAdenosine triphosphateC10H16N5O13P32
91 mMTCEPC9H15O6P2
101 mMMagnesium chlorideMgCl22
Specimen

Experiment-ID: 1 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES

IDConc. (mg/ml)Details
11.5Specimen was imaged at a 40 degree tilt.
20.2Sample was applied to holey C-flat grids, which previously had thin carbon floated on them. Grids were also pretreated with 0.1 % (w/v) poly-L-lysine hydrobromine (Polysciences).
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid typeDetails
11GOLD300UltrAuFoil R1.2/1.3
22C-flatSample was applied to holey C-flat grids, which previously had thin carbon floated on them. Grids were also pretreated with 0.1 % (w/v) poly-L-lysine hydrobromine (Polysciences).
Vitrification

Chamber temperature: 277.15 K / Cryogen name: ETHANE / 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. / Entry-ID: 7S66 / Humidity: 95 % / Instrument: HOMEMADE PLUNGER

IDSpecimen-ID
11
22

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 200 kV / Alignment procedure: COMA FREE / C2 aperture diameter: 70 µm / Calibrated magnification: 43478 X / Cryogen: NITROGEN / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TALOS ARCTICA / Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 36000 X / Temperature (max): 70 K / Temperature (min): 70 K / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

IDCalibrated defocus max (nm)Calibrated defocus min (nm)DetailsNominal defocus max (nm)Nominal defocus min (nm)Specimen-ID
1-1700-800Sample was imaged at a 40 degree tilt.-1700-8001
2Sample was imaged at 0 degree tilt.1500-5002
Image recording

Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Num. of real imagesDetails
118481137Tilted dataset
229518405Thin carbon dataset
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 36 / Used frames/image: 1-36

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Processing

EM software
IDNameVersionCategoryImaging-ID
3Leginonimage acquisition1
5RELION3CTF correction
8UCSF Chimeramodel fitting
10Cootmodel refinement
14Leginonimage acquisition2
15RELION3initial Euler assignment
17RELION3final Euler assignment
19RELION3classification
21RELION33D reconstruction
CTF correctionDetails: CTF Refinement performed in Relion / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2016826
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91869 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3K0C

3k0c

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