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- EMDB-24852: Extended conformation of daytime state KaiC -

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Basic information

Entry
Database: EMDB / ID: EMD-24852
TitleExtended conformation of daytime state KaiC
Map dataSharpened map
Sample
  • Complex: Compressed state hexamer of nighttime state phosphomutant KaiC (KaiC-EA)
    • Protein or peptide: Circadian clock protein kinase KaiC
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
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
Circadian clock oscillator protein KaiC
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSandate CR / Swan JA / Partch CL / Lander GC
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7s67
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24852.png

Downloads & links

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Map

FileDownload / File: emd_24852.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.029946547 - 0.04246971
Average (Standard dev.)-3.421153e-06 (±0.001973754)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 220.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ720720720
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0300.042-0.000

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Supplemental data

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Additional map: Unsharpened map

Fileemd_24852_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_24852_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_24852_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Compressed state hexamer of nighttime state phosphomutant KaiC (K...

EntireName: Compressed state hexamer of nighttime state phosphomutant KaiC (KaiC-EA)
Components
  • Complex: Compressed state hexamer of nighttime state phosphomutant KaiC (KaiC-EA)
    • Protein or peptide: Circadian clock protein kinase KaiC
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Compressed state hexamer of nighttime state phosphomutant KaiC (K...

SupramoleculeName: Compressed state hexamer of nighttime state phosphomutant KaiC (KaiC-EA)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Synechococcus elongatus (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Recombinant plasmid: pET-28b
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Circadian clock protein kinase KaiC

MacromoleculeName: Circadian clock protein kinase KaiC / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Synechococcus elongatus (bacteria)
Molecular weightTheoretical: 58.084781 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MTSAEMTSPN NNSEHQAIAK MRTMIEGFDD ISHGGLPIGR STLVSGTSGT GKTLFSIQFL YNGIIEFDEP GVFVTFEETP QDIIKNARS FGWDLAKLVD EGKLFILDAS PDPEGQEVVG GFDLSALIER INYAIQKYRA RRVSIDSVTS VFQQYDASSV V RRELFRLV ...String:
MTSAEMTSPN NNSEHQAIAK MRTMIEGFDD ISHGGLPIGR STLVSGTSGT GKTLFSIQFL YNGIIEFDEP GVFVTFEETP QDIIKNARS FGWDLAKLVD EGKLFILDAS PDPEGQEVVG GFDLSALIER INYAIQKYRA RRVSIDSVTS VFQQYDASSV V RRELFRLV ARLKQIGATT VMTTERIEEY GPIARYGVEE FVSDNVVILR NVLEGERRRR TLEILKLRGT SHMKGEYPFT IT DHGINIF PLGAMRLTQR SSNVRVSSGV VRLDEMCGGG FFKDSIILAT GATGTGKTLL VSRFVENACA NKERAILFAY EES RAQLLR NAYSWGMDFE EMERQNLLKI VCAYPESAGL EDHLQIIKSE INDFKPARIA IDSLSALARG VSNNAFRQFV IGVT GYAKQ EEITGLFTNT SDQFMGAHSI TDSHIAEITD TIILLQYVEI RGEMSRAINV FKMRGSWHDK AIREFMISDK GPDIK DSFR NFERIISGSP TRITVDEKSE LSRIVRGVQE KGPES

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 6 / 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 #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
150.0 mMNaClSodium chlorideSodium chloride
1.0 mMC10H16N5O13P3Adenosine triphosphate
1.0 mMMgCl2Magnesium chloride
1.0 mMC9H15O6PTCEP
4.0 mMC13H17F13NO4PFluorinated fos-choline-8
GridModel: UltrAuFoil R1.2/1.3 / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS
Details: Graphene-coated grids were made hydrophilic via treatment by UV/ozone cleaner.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
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..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: -2.0 µm / Calibrated defocus min: -0.5 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-62 / Number grids imaged: 1 / Number real images: 1541 / Average exposure time: 6.2 sec. / Average electron dose: 40.3 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 427620
CTF correctionSoftware - Name: RELION (ver. 3.0) / Details: CTF Refinement performed in Relion
Startup modelType of model: NONE
Details: Ab initio reconstruction was generated using cryoSPARC version 2.0
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 89892
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3k0c

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7s67:
Extended conformation of daytime state KaiC

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