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- EMDB-5381: Cdc6-induced Conformational Changes in ORC Bound To Origin DNA Re... -

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Basic information

Entry
Database: EMDB / ID: EMD-5381
TitleCdc6-induced Conformational Changes in ORC Bound To Origin DNA Revealed by Cryo-Electron Microscopy. This map may be mirrored (based on comparison to EMD-5625).
Map datacryoEM structure of Yeast Origin Recognition complex with dsDNA and Cdc6. This map may be mirrored (based on comparison to EMD-5625).
Sample
  • Sample: Yeast Origin Recognition Complex with dsDNA and Cdc6
  • Protein or peptide: Yeast Origin Recognition Complex and Cdc6
KeywordsORC / Cdc6
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsSun J / Kawakami H / Zech J / Speck C / Stillman B / Li H
CitationJournal: Structure / Year: 2012
Title: Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy.
Authors: Jingchuan Sun / Hironori Kawakami / Juergen Zech / Christian Speck / Bruce Stillman / Huilin Li /
Abstract: The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of ...The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.
History
DepositionJan 8, 2012-
Header (metadata) releaseJan 11, 2012-
Map releaseMar 14, 2012-
UpdateJun 4, 2014-
Current statusJun 4, 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
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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_5381_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5381.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM structure of Yeast Origin Recognition complex with dsDNA and Cdc6. This map may be mirrored (based on comparison to EMD-5625).
Voxel sizeX=Y=Z: 4.23 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.47482175 - 7.11127949
Average (Standard dev.)0.21266665 (±0.72353882)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-24-24
Dimensions484848
Spacing484848
CellA=B=C: 203.04001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.234.234.23
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z203.040203.040203.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS484848
D min/max/mean-0.4757.1110.213

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Supplemental data

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Sample components

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Entire : Yeast Origin Recognition Complex with dsDNA and Cdc6

EntireName: Yeast Origin Recognition Complex with dsDNA and Cdc6
Components
  • Sample: Yeast Origin Recognition Complex with dsDNA and Cdc6
  • Protein or peptide: Yeast Origin Recognition Complex and Cdc6

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Supramolecule #1000: Yeast Origin Recognition Complex with dsDNA and Cdc6

SupramoleculeName: Yeast Origin Recognition Complex with dsDNA and Cdc6 / type: sample / ID: 1000
Details: ORC and Cdc6 are mixed with 66 bp DNA with ARS1 in ATPrS
Number unique components: 8
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa

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Macromolecule #1: Yeast Origin Recognition Complex and Cdc6

MacromoleculeName: Yeast Origin Recognition Complex and Cdc6 / type: protein_or_peptide / ID: 1 / Name.synonym: ORC, Cdc6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: ORC, Cdc6
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pCITE-2a

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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

Concentration0.4 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES/KOH, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 mM KGlu
GridDetails: 300 mesh lacey grid with thin continuous carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: OTHER / Details: Vitrification instrument: FEI Vitrobot / Method: Blot 5 seconds

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Gatan 626200 / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON COOLSCAN / Digitization - Sampling interval: 6.35 µm / Average electron dose: 15 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each particle set
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1.8 / Number images used: 54000

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