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- EMDB-5625: Architecture of a helicase loading intermediate containing ORC-Cd... -

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Basic information

Entry
Database: EMDB / ID: EMD-5625
TitleArchitecture of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 on DNA reveals similarity to DNA polymerase clamp loading complexes
Map dataReconstruction of ORC-Cdc6-Cdt1-Mcm2-7 complex at DNA origin in ATPrS
Sample
  • Sample: ORC-Cdc6-Cdt1-Mcm2-7 complex
  • Protein or peptide: ORC-Cdc6-Cdt1-Mcm2-7 complex
KeywordsPreRC / ORC / Cdc6 / Cdt1 / Mcm2-7
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsSun J / Evrin C / Samel S / Fernandez-Cid A / Riera A / Kawakami H / Stillman B / Speck C / Li H
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.
Authors: Jingchuan Sun / Cecile Evrin / Stefan A Samel / Alejandra Fernández-Cid / Alberto Riera / Hironori Kawakami / Bruce Stillman / Christian Speck / Huilin Li /
Abstract: In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using ...In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.
History
DepositionApr 4, 2013-
Header (metadata) releaseJul 3, 2013-
Map releaseJul 24, 2013-
UpdateAug 14, 2013-
Current statusAug 14, 2013Processing 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
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_5625.map.gz / Format: CCP4 / Size: 670.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of ORC-Cdc6-Cdt1-Mcm2-7 complex at DNA origin in ATPrS
Voxel sizeX=Y=Z: 4.23 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.49997807 - 7.94068527
Average (Standard dev.)0.25216204 (±0.86932105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-28-28-28
Dimensions565656
Spacing565656
CellA=B=C: 236.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.234.234.23
M x/y/z565656
origin x/y/z0.0000.0000.000
length x/y/z236.880236.880236.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-28-28-28
NC/NR/NS565656
D min/max/mean-1.5007.9410.252

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Supplemental data

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Sample components

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Entire : ORC-Cdc6-Cdt1-Mcm2-7 complex

EntireName: ORC-Cdc6-Cdt1-Mcm2-7 complex
Components
  • Sample: ORC-Cdc6-Cdt1-Mcm2-7 complex
  • Protein or peptide: ORC-Cdc6-Cdt1-Mcm2-7 complex

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Supramolecule #1000: ORC-Cdc6-Cdt1-Mcm2-7 complex

SupramoleculeName: ORC-Cdc6-Cdt1-Mcm2-7 complex / type: sample / ID: 1000 / Number unique components: 14
Molecular weightTheoretical: 1.1 MDa

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Macromolecule #1: ORC-Cdc6-Cdt1-Mcm2-7 complex

MacromoleculeName: ORC-Cdc6-Cdt1-Mcm2-7 complex / type: protein_or_peptide / ID: 1 / Name.synonym: OCCM / Number of copies: 1 / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 1.1 MDa

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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

Concentration0.1 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES-KOH
GridDetails: thin carbon layer on holey carbon, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK I
Method: A thin carbon layer was floated onto holey carbon and glow discharged. 2.8 uL sample was added and after 30 seconds was blotted for 5 seconds and plunge-frozen.

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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: 4.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 60000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 400K magnification.
DateMar 8, 2010
Image recordingCategory: FILM / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 800 / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: each particle set by ctfit
Final two d classificationNumber classes: 1104
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1, EMAN2 / Number images used: 92049
DetailsParticles were selected with e2boxer.py in swarm mode. Additional good particles were selected and bad particles were removed manually. After structural factor and CTF correction in EMAN1, the phase-flipped particles were normalized (edgenorm, hp=1) and pooled. Refine2d.py was run and particles in bad class averages were removed. Initial models were made using EMAN2, but 3D refinement was performed using EMAN1.8 without ctfc or ctfcw.

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