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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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5625_1.png

Downloads & links

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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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.23 Å/pix.
x 56 pix.
= 236.88 Å		4.23 Å/pix.
x 56 pix.
= 236.88 Å		4.23 Å/pix.
x 56 pix.
= 236.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.23 Å
Density

Histogram

Histogram (log scale)
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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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

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Image processing

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.
CTF correctionDetails: each particle set by ctfit
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
Final two d classificationNumber classes: 1104

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