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- EMDB-5857: Architectures of Mcm2-7 Double Hexamer Assembly Intermediates Rev... -

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Basic information

Entry
Database: EMDB / ID: EMD-5857
TitleArchitectures of Mcm2-7 Double Hexamer Assembly Intermediates Reveal Helicase Loading Mechanism
Map data3D reconstruction of Mcm2-7 double hexamer
Sample
  • Sample: Mcm2-7 double hexamer from Saccharomyces cerevisiae
  • Protein or peptide: Mini-Chromosome MaintenanceMinichromosome maintenance
KeywordsMcm2-7 double hexamer / MBP fusion / Helicase loading / 3DEM
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 15.0 Å
AuthorsSun J / Fernandez-Cid A / Riera A / Stillman B / Speck C / Li H
CitationJournal: Genes Dev / Year: 2014
Title: Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function.
Authors: Jingchuan Sun / Alejandra Fernandez-Cid / Alberto Riera / Silvia Tognetti / Zuanning Yuan / Bruce Stillman / Christian Speck / Huilin Li /
Abstract: Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S ...Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
History
DepositionJan 9, 2014-
Header (metadata) releaseMar 5, 2014-
Map releaseNov 12, 2014-
UpdateNov 12, 2014-
Current statusNov 12, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5857.jpg

Downloads & links

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Map

FileDownload / File: emd_5857.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of Mcm2-7 double hexamer
Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.26576003 - 4.10939789
Average (Standard dev.)0.09086699 (±0.3930566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 339.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z339.200339.200339.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.2664.1090.091

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Supplemental data

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Sample components

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Entire : Mcm2-7 double hexamer from Saccharomyces cerevisiae

EntireName: Mcm2-7 double hexamer from Saccharomyces cerevisiae
Components
  • Sample: Mcm2-7 double hexamer from Saccharomyces cerevisiae
  • Protein or peptide: Mini-Chromosome MaintenanceMinichromosome maintenance

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Supramolecule #1000: Mcm2-7 double hexamer from Saccharomyces cerevisiae

SupramoleculeName: Mcm2-7 double hexamer from Saccharomyces cerevisiae / type: sample / ID: 1000 / Oligomeric state: dimer of hexamers / Number unique components: 1
Molecular weightExperimental: 1.2 MDa / Theoretical: 1.2 MDa / Method: Gel filtration

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Macromolecule #1: Mini-Chromosome Maintenance

MacromoleculeName: Mini-Chromosome Maintenance / type: protein_or_peptide / ID: 1 / Name.synonym: Mcm / Number of copies: 2 / Oligomeric state: dimer of hexamers / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE / Details: 1% uranyl acetate
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK I / Method: Blot 5 seconds

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateOct 10, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 100 / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: each particle set
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: EMAN1.8 / Number images used: 22630
DetailsParticles were picked manually.

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Atomic model buiding 1

Initial modelPDB ID:

3f9v

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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