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- EMDB-1157: ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. -

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Basic information

Entry
Database: EMDB / ID: EMD-1157
TitleATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.
Map data3D map of the yeast (Saccharomyces cerevisiae)DNA origin recognition complex (ORC) bound with initiation factor Cdc6p.
Sample
  • Sample: Yeast Origin Recognition complex and Cdc6p
  • Protein or peptide: Orc1p
  • Protein or peptide: Orc2p
  • Protein or peptide: Orc3p
  • Protein or peptide: Orc4p
  • Protein or peptide: Orc5p
  • Protein or peptide: Orc6p
  • Protein or peptide: Cdc6p
Function / homologyOrigin recognition complex, subunit 2 / nuclear origin of replication recognition complex / nuclear pre-replicative complex / AAA+ ATPase domain
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsSpeck C / Chen Z / Li H / Stillman B
CitationJournal: Nat Struct Mol Biol / Year: 2005
Title: ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.
Authors: Christian Speck / Zhiqiang Chen / Huilin Li / Bruce Stillman /
Abstract: Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences ...Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.
History
DepositionSep 7, 2005-
Header (metadata) releaseSep 7, 2005-
Map releaseSep 7, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.946640809
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.946640809
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1157.gif

Downloads & links

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Map

FileDownload / File: emd_1157.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of the yeast (Saccharomyces cerevisiae)DNA origin recognition complex (ORC) bound with initiation factor Cdc6p.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 100 pix.
= 254. Å		2.54 Å/pix.
x 100 pix.
= 254. Å		2.54 Å/pix.
x 100 pix.
= 254. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.54 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.359 / Movie #1: 0.9466408
Minimum - Maximum0.00192997 - 5.82841
Average (Standard dev.)0.101892 (±0.462275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 254 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z254.000254.000254.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-70-70-69
NX/NY/NZ140140140
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean0.0025.8280.102

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Supplemental data

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Sample components

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Entire : Yeast Origin Recognition complex and Cdc6p

EntireName: Yeast Origin Recognition complex and Cdc6p
Components
  • Sample: Yeast Origin Recognition complex and Cdc6p
  • Protein or peptide: Orc1p
  • Protein or peptide: Orc2p
  • Protein or peptide: Orc3p
  • Protein or peptide: Orc4p
  • Protein or peptide: Orc5p
  • Protein or peptide: Orc6p
  • Protein or peptide: Cdc6p

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Supramolecule #1000: Yeast Origin Recognition complex and Cdc6p

SupramoleculeName: Yeast Origin Recognition complex and Cdc6p / type: sample / ID: 1000 / Details: highly purified, monodisperse / Oligomeric state: hetero-heptamer / Number unique components: 7
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa / Method: sedimentation

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Macromolecule #1: Orc1p

MacromoleculeName: Orc1p / type: protein_or_peptide / ID: 1 / Name.synonym: ORC subunit 1 / Details: largest subunit, ATPase / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculovirus bvORC1:6
SequenceGO: nuclear origin of replication recognition complex / InterPro: AAA+ ATPase domain

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Macromolecule #2: Orc2p

MacromoleculeName: Orc2p / type: protein_or_peptide / ID: 2 / Name.synonym: ORC subunit 2 / Details: The second subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 73 KDa / Theoretical: 73 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC2:5
SequenceGO: nuclear origin of replication recognition complex / InterPro: Origin recognition complex, subunit 2

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Macromolecule #3: Orc3p

MacromoleculeName: Orc3p / type: protein_or_peptide / ID: 3 / Name.synonym: ORC subunit 3 / Details: The third subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 72 KDa / Theoretical: 72 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC3:4
SequenceGO: nuclear origin of replication recognition complex

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Macromolecule #4: Orc4p

MacromoleculeName: Orc4p / type: protein_or_peptide / ID: 4 / Name.synonym: ORC subunit 4 / Details: The forth subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 61 KDa / Theoretical: 61 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC3:4
SequenceGO: nuclear origin of replication recognition complex

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Macromolecule #5: Orc5p

MacromoleculeName: Orc5p / type: protein_or_peptide / ID: 5 / Name.synonym: ORC subunit 5 / Details: The fifth subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 53 KDa / Theoretical: 53 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC2:5
SequenceGO: nuclear origin of replication recognition complex

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Macromolecule #6: Orc6p

MacromoleculeName: Orc6p / type: protein_or_peptide / ID: 6 / Name.synonym: ORC subunit 6 / Details: The sixth subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Hi-5 cell / Recombinant plasmid: baculovirus bvORC1:6
SequenceGO: nuclear origin of replication recognition complex

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Macromolecule #7: Cdc6p

MacromoleculeName: Cdc6p / type: protein_or_peptide / ID: 7 / Name.synonym: Cdc6 / Details: initiation factor / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pGex-6P1-CDC6
SequenceGO: nuclear pre-replicative complex

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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

Concentration0.02 mg/mL
BufferpH: 7.5 / Details: 50mM HEPES-KOH, 100mM KCl, 1mM EDTA, 1mM EGTA
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 30 seconds.
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 1200EX
Alignment procedureLegacy - Astigmatism: corrected at 250,000 X
DateOct 1, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 12.7 µm / Number real images: 50 / Average electron dose: 10 e/Å2 / Od range: 1.5 / Bits/pixel: 14
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 5.6 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER / Tilt angle max: 45

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Image processing

CTF correctionDetails: each film
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, EMAN / Number images used: 12000
Final two d classificationNumber classes: 50

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Atomic model buiding 1

Detailsmanual fit using program O
RefinementProtocol: RIGID BODY FIT

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