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- PDB-5n8y: KaiCBA circadian clock backbone model based on a Cryo-EM density -

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Basic information

Entry
Database: PDB / ID: 5n8y
TitleKaiCBA circadian clock backbone model based on a Cryo-EM density
Components
  • Circadian clock protein KaiA
  • Circadian clock protein KaiB
  • Circadian clock protein kinase KaiC
KeywordsTRANSFERASE / AAA+-ATPase / Kinase / Circadian Clock Complex / Cyanobacteria / Fold-switch
Function / homology
Function and homology information


detection of redox state / negative regulation of protein dephosphorylation / regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / negative regulation of phosphorylation / positive regulation of circadian rhythm / protein serine/threonine/tyrosine kinase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / regulation of circadian rhythm ...detection of redox state / negative regulation of protein dephosphorylation / regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / negative regulation of phosphorylation / positive regulation of circadian rhythm / protein serine/threonine/tyrosine kinase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / regulation of circadian rhythm / 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 / plasma membrane / cytoplasm
Similarity search - Function
Circadian clock protein KaiA, N-terminal / Circadian clock protein KaiA, N-terminal domain / KaiA N-terminal domain profile. / Circadian clock protein KaiA / Circadian clock protein KaiB / Circadian clock protein KaiA, C-terminal / KaiA C-terminal domain / KaiA C-terminal domain profile. / KaiA / Circadian clock protein KaiB-like ...Circadian clock protein KaiA, N-terminal / Circadian clock protein KaiA, N-terminal domain / KaiA N-terminal domain profile. / Circadian clock protein KaiA / Circadian clock protein KaiB / Circadian clock protein KaiA, C-terminal / KaiA C-terminal domain / KaiA C-terminal domain profile. / KaiA / Circadian clock protein KaiB-like / KaiA/RbsU helical domain superfamily / KaiB domain / KaiB domain / KaiB / Circadian clock KaiC, bacteria / : / Circadian clock protein kinase KaiC / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / CheY-like superfamily / Thioredoxin-like superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Circadian clock oscillator protein KaiC / Circadian clock oscillator protein KaiB / Circadian clock oscillator protein KaiA
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsSchuller, J.M. / Snijder, J. / Loessl, P. / Heck, A.J.R. / Foerster, F.
CitationJournal: Science / Year: 2017
Title: Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state.
Authors: Joost Snijder / Jan M Schuller / Anika Wiegard / Philip Lössl / Nicolas Schmelling / Ilka M Axmann / Jürgen M Plitzko / Friedrich Förster / Albert J R Heck /
Abstract: 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 ...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 5'-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.
History
DepositionFeb 24, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 29, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.details / _em_software.name
Revision 1.2Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: em_software / pdbx_unobs_or_zero_occ_atoms ...em_software / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Item: _em_software.name
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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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
G: Circadian clock protein KaiB
H: Circadian clock protein KaiB
K: Circadian clock protein KaiB
J: Circadian clock protein KaiB
I: Circadian clock protein KaiB
L: Circadian clock protein KaiB
M: Circadian clock protein KaiA
N: Circadian clock protein KaiA
O: Circadian clock protein KaiA
P: Circadian clock protein KaiA
Q: Circadian clock protein KaiA
R: Circadian clock protein KaiA
S: Circadian clock protein KaiA
T: Circadian clock protein KaiA
U: Circadian clock protein KaiA
V: Circadian clock protein KaiA
W: Circadian clock protein KaiA
X: Circadian clock protein KaiA


Theoretical massNumber of molelcules
Total (without water)809,13324
Polymers809,13324
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, KaiC6B6A12 - native mass spec
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Circadian clock protein kinase KaiC /


Mass: 58072.773 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: kaiC, Synpcc7942_1216, see0011 / Production host: Escherichia coli (E. coli)
References: UniProt: Q79PF4, non-specific serine/threonine protein kinase
#2: Protein
Circadian clock protein KaiB /


Mass: 11450.387 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: kaiB, Synpcc7942_1217, see0010 / Production host: Escherichia coli (E. coli) / References: UniProt: Q79PF5
#3: Protein
Circadian clock protein KaiA /


Mass: 32666.199 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: kaiA, Synpcc7942_1218, see0009 / Production host: Escherichia coli (E. coli) / References: UniProt: Q79PF6

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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: KaiCBA circadian clock complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Synechococcus elongatus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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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 / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15.2 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1TOM Toolboxparticle selectionTOM picker
4CTFFIND4CTF correction
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32498 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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