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- EMDB-5672: CryoEM Structure of the KaiBC Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5672
TitleCryoEM Structure of the KaiBC Complex
Map dataReconstruction of KaiB-KaiC(489 deletion)
Sample
  • Sample: KaiB in complex with KaiC(489del)
  • Protein or peptide: KaiB
  • Protein or peptide: KaiC
KeywordsCircadian oscillator / cryoEM / MDFF / protein-protein interface / Synechococcus elongatus
Function / homology
Function and homology information


: / : / regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / negative regulation of phosphorylation / 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 ...: / : / regulation of phosphorelay signal transduction system / negative regulation of circadian rhythm / entrainment of circadian clock / negative regulation of phosphorylation / 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 KaiB / Circadian clock protein KaiB / Circadian clock protein KaiB-like / KaiB domain / KaiB domain / KaiB / Circadian clock KaiC, bacteria / Circadian clock KaiC, bacteria / : / Circadian clock protein kinase KaiC ...Circadian clock protein KaiB / Circadian clock protein KaiB / Circadian clock protein KaiB-like / KaiB domain / KaiB domain / KaiB / Circadian clock KaiC, bacteria / Circadian clock KaiC, bacteria / : / Circadian clock protein kinase KaiC / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / Thioredoxin-like superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Circadian clock oscillator protein KaiC / Circadian clock oscillator protein KaiB
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsVillarreal SA / Pattanayek R / Williams DR / Mori T / Qin X / Johnson CH / Egli M / Stewart PL
CitationJournal: J Mol Biol / Year: 2013
Title: CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates that KaiB monomers interact with KaiC and block ATP binding clefts.
Authors: Seth A Villarreal / Rekha Pattanayek / Dewight R Williams / Tetsuya Mori / Ximing Qin / Carl H Johnson / Martin Egli / Phoebe L Stewart /
Abstract: The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote ...The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB-KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.
History
DepositionMay 16, 2013-
Header (metadata) releaseJun 26, 2013-
Map releaseJul 3, 2013-
UpdateSep 4, 2013-
Current statusSep 4, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0877
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0877
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5672_1.tif

Downloads & links

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Map

FileDownload / File: emd_5672.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of KaiB-KaiC(489 deletion)
Voxel sizeX=Y=Z: 2.32 Å
Density
Contour LevelBy AUTHOR: 0.0877 / Movie #1: 0.0877
Minimum - Maximum-0.10229599 - 0.245758
Average (Standard dev.)0.0020918 (±0.04392396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin404040
Dimensions808080
Spacing808080
CellA=B=C: 185.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.322.322.32
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z185.600185.600185.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS404040
NC/NR/NS808080
D min/max/mean-0.1020.2460.002

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Supplemental data

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Sample components

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Entire : KaiB in complex with KaiC(489del)

EntireName: KaiB in complex with KaiC(489del)
Components
  • Sample: KaiB in complex with KaiC(489del)
  • Protein or peptide: KaiB
  • Protein or peptide: KaiC

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Supramolecule #1000: KaiB in complex with KaiC(489del)

SupramoleculeName: KaiB in complex with KaiC(489del) / type: sample / ID: 1000
Details: Visualization of the sample showed complexes of KaiBC and KaiC.
Oligomeric state: One Hexamer of KaiC binds to 6 monomers of KaiB
Number unique components: 2
Molecular weightTheoretical: 510 KDa

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Macromolecule #1: KaiB

MacromoleculeName: KaiB / type: protein_or_peptide / ID: 1 / Name.synonym: Circadian clock protein KaiB / Number of copies: 6 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Synechococcus elongatus (bacteria) / Strain: PCC 7942 / Location in cell: Cytoplasm
Molecular weightTheoretical: 12 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Circadian clock oscillator protein KaiB / GO: GO: 3773505 / InterPro: Circadian clock protein KaiB

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Macromolecule #2: KaiC

MacromoleculeName: KaiC / type: protein_or_peptide / ID: 2 / Name.synonym: Circadian clock protein kinase KaiC / Details: C terminal truncation of KaiC at 489 / Number of copies: 1 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Synechococcus elongatus (bacteria) / Strain: PCC 7942 / Location in cell: Cytoplasm
Molecular weightTheoretical: 340 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Circadian clock oscillator protein KaiC / GO: GO: 3773504 / InterPro: Circadian clock KaiC, bacteria

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferDetails: 20 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA
GridDetails: homemade holey carbon film and C-flat grids (Protochips, Inc.)
VitrificationCryogen name: ETHANE / Chamber temperature: 184 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 254669 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: -5.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 200000
Sample stageSpecimen holder model: OTHER
TemperatureMin: 98 K / Max: 105 K / Average: 101 K
DateJun 2, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 11734 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 2000
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF
Software - Name: FREALIGN, Rubinstein_2_Refinement_package, IMAGIC
Details: Final map was calculated from 3 data sets. / Number images used: 195226
DetailsThe particles were selected manually.

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