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- EMDB-29506: Structure of dodecameric KaiC-RS-S413E/S414E complexed with KaiB-... -

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Basic information

Entry
Database: EMDB / ID: EMD-29506
TitleStructure of dodecameric KaiC-RS-S413E/S414E complexed with KaiB-RS solved by cryo-EM
Map dataComposite map of two hexamer reconstructions aligned and combined by taking the maximum absolute value
Sample
  • Complex: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS
    • Protein or peptide: Circadian clock protein KaiC
    • Protein or peptide: Circadian clock protein KaiB
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
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
Circadian clock protein KaiB / non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesCereibacter sphaeroides (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsPadua RAP / Grant T / Pitsawong W / Hoemberger MS / Otten R / Bradshaw N / Grigorieff N / Kern D
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29506.png

Downloads & links

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Map

FileDownload / File: emd_29506.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of two hexamer reconstructions aligned and combined by taking the maximum absolute value
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.5405234 - 1.1670166
Average (Standard dev.)0.0025584998 (±0.029546771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 512.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS

EntireName: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS
Components
  • Complex: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS
    • Protein or peptide: Circadian clock protein KaiC
    • Protein or peptide: Circadian clock protein KaiB
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS

SupramoleculeName: Dodecamer of KaiC-RS-S413E/S414E bound with KaiC-RS / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Molecular weightTheoretical: 875 KDa

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Macromolecule #1: Circadian clock protein KaiC

MacromoleculeName: Circadian clock protein KaiC / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Molecular weightTheoretical: 62.666133 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAMGIGKSPT GIQGFDELTL GGLPTGRPSL VCGSAGCGKT LFASTFLING VRDHGEPGVF VTFEERPEDI VNNVASLGFE LDKLIEEEK IAIEHIAVDP SEVAEIGDYD LEGLFLRLEL AIDTVGAKRV VLDTIESLFS AFSNPAILRA EIRRLFDWLK E RGLTTVIT ...String:
GAMGIGKSPT GIQGFDELTL GGLPTGRPSL VCGSAGCGKT LFASTFLING VRDHGEPGVF VTFEERPEDI VNNVASLGFE LDKLIEEEK IAIEHIAVDP SEVAEIGDYD LEGLFLRLEL AIDTVGAKRV VLDTIESLFS AFSNPAILRA EIRRLFDWLK E RGLTTVIT AERGDGALTR QGLEEYVSDC VILLDHRVEN QISTRRLRIV KYRGTAHGTN EYPFLIDTDG FSVLPVSALG LL HQVHEER IASGVPDLDA MMAGGGFFRG SSILVSGVAG AGKSSLAAHF AAAACARGER AMYFSFEEAA DQAVRNMRSL GLD LGRWRD AGLLRFMATR PTFYSLEMHL AVILREVMRF EPSVVVLDPI SAFTESGDRL EVQSMLLRIV DFLKNRGITG IFTH LAHSQ NEATTDAGLE ELMDGWVLML NREVNGEFNR ELYLLKARGM AHSNQVREFL MSDRGISLLP PHLGEGGALT GTARK AEEA RLRRAEIERQ TELGRLQQQI EQRRRRARAQ IEALEAELQA EEIALKALVE SESAHERQRL ADADTLARSR GNERFA DLL MNKGE

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Macromolecule #2: Circadian clock protein KaiB

MacromoleculeName: Circadian clock protein KaiB / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Molecular weightTheoretical: 10.317127 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GAMGRRLVLY VAGQTPKSLA AISNLRRICE ENLPGQYEVE VIDLKQNPRL AKEHSIVAIP TLVRELPVPI RKIIGDLSDK EQVLVNLKM DME

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 24 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 48 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 6.5
GridModel: C-flat-1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K2 BASE (4k x 4k) / Average electron dose: 100.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0-alpha)
Final 3D classificationSoftware - Name: cisTEM (ver. 2.0.0-alpha)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0-alpha)
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 2.0.0-alpha) / Number images used: 160000

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8fwj:
Structure of dodecameric KaiC-RS-S413E/S414E complexed with KaiB-RS solved by cryo-EM

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