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- EMDB-29508: Structure of dodecameric KaiC-RS-S413E/S414E with bound KaiB-RS -... -

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Basic information

Entry
Database: EMDB / ID: EMD-29508
TitleStructure of dodecameric KaiC-RS-S413E/S414E with bound KaiB-RS - single hexamer focus map
Map dataKaiC-RS-S413E/S414E with bound KaiB-RS sharpened map with refinement focused on a single hexamer
Sample
  • Complex: Dodecamer of KaiC-RS-S413E/S414E
    • Protein or peptide: KaiC-RS-S413E/S414E
    • Protein or peptide: KaiB-RS
Keywordsautokinase / CIRCADIAN CLOCK PROTEIN
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) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29508.png

Downloads & links

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Map

FileDownload / File: emd_29508.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKaiC-RS-S413E/S414E with bound KaiB-RS sharpened map with refinement focused on a single hexamer
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 432 pix.
= 432. Å		1 Å/pix.
x 432 pix.
= 432. Å		1 Å/pix.
x 432 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.62181836 - 1.3145145
Average (Standard dev.)0.0027943887 (±0.036103234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 432.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: unsharpened half map

Fileemd_29508_half_map_1.map
Annotationunsharpened half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unsharpened half map

Fileemd_29508_half_map_2.map
Annotationunsharpened half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dodecamer of KaiC-RS-S413E/S414E

EntireName: Dodecamer of KaiC-RS-S413E/S414E
Components
  • Complex: Dodecamer of KaiC-RS-S413E/S414E
    • Protein or peptide: KaiC-RS-S413E/S414E
    • Protein or peptide: KaiB-RS

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

SupramoleculeName: Dodecamer of KaiC-RS-S413E/S414E / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Molecular weightTheoretical: 751 KDa

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Macromolecule #1: KaiC-RS-S413E/S414E

MacromoleculeName: KaiC-RS-S413E/S414E / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAMGIGKSPT GIQGFDELTL GGLPTGRPSL VCGSAGCGKT LFASTFLING VRDHGEPGVF VTFEERPEDI VNNVASLGFE LDKLIEEEKI AIEHIAVDPS EVAEIGDYDL EGLFLRLELA IDTVGAKRVV LDTIESLFSA FSNPAILRAE IRRLFDWLKE RGLTTVITAE ...String:
GAMGIGKSPT GIQGFDELTL GGLPTGRPSL VCGSAGCGKT LFASTFLING VRDHGEPGVF VTFEERPEDI VNNVASLGFE LDKLIEEEKI AIEHIAVDPS EVAEIGDYDL EGLFLRLELA IDTVGAKRVV LDTIESLFSA FSNPAILRAE IRRLFDWLKE RGLTTVITAE RGDGALTRQG LEEYVSDCVI LLDHRVENQI STRRLRIVKY RGTAHGTNEY PFLIDTDGFS VLPVSALGLL HQVHEERIAS GVPDLDAMMA GGGFFRGSSI LVSGVAGAGK SSLAAHFAAA ACARGERAMY FSFEEAADQA VRNMRSLGLD LGRWRDAGLL RFMATRPTFY SLEMHLAVIL REVMRFEPSV VVLDPISAFT ESGDRLEVQS MLLRIVDFLK NRGITGIFTH LAHSQNEATT DAGLEELMDG WVLMLNREVN GEFNRELYLL KARGMAHSNQ VREFLMSDRG ISLLPPHLGE GGALTGTARK AEEARLRRAE IERQTELGRL QQQIEQRRRR ARAQIEALEA ELQAEEIALK ALVESESAHE RQRLADADTL ARSRGNERFA DLLMNKGE

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Macromolecule #2: KaiB-RS

MacromoleculeName: KaiB-RS / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Cereibacter sphaeroides (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GAMGRRLVLY VAGQTPKSLA AISNLRRICE ENLPGQYEVE VIDLKQNPRL AKEHSIVAIP TLVRELPVPI RKIIGDLSDK EQVLVNLKMD ME

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

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

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Image processing

Startup modelType of model: NONE
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
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0-alpha)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0-alpha)
Final 3D classificationSoftware - Name: cisTEM (ver. 2.0.0-alpha)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT

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