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Yorodumi- EMDB-1156: ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1156 | |||||||||
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Title | ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. | |||||||||
Map data | 3D maps of the yeast (Saccharomyces cerevisiae) DNA origin recognition complex (ORC) and the ORC in complex with the replication initiator Cdc6. | |||||||||
Sample |
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Function / homology | Origin recognition complex, subunit 2 / nuclear origin of replication recognition complex / AAA+ ATPase domain Function and homology information | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
Authors | Speck C / Chen Z / Li H / Stillman B | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1156.map.gz | 252.1 KB | EMDB map data format | |
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Header (meta data) | emd-1156-v30.xml emd-1156.xml | 15.9 KB 15.9 KB | Display Display | EMDB header |
Images | 1156.gif | 30.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1156 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1156 | HTTPS FTP |
-Validation report
Summary document | emd_1156_validation.pdf.gz | 193 KB | Display | EMDB validaton report |
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Full document | emd_1156_full_validation.pdf.gz | 192.1 KB | Display | |
Data in XML | emd_1156_validation.xml.gz | 5.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1156 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1156 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1156.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D maps of the yeast (Saccharomyces cerevisiae) DNA origin recognition complex (ORC) and the ORC in complex with the replication initiator Cdc6. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.54 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Yeast Origin Recognition complex
Entire | Name: Yeast Origin Recognition complex |
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Components |
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-Supramolecule #1000: Yeast Origin Recognition complex
Supramolecule | Name: Yeast Origin Recognition complex / type: sample / ID: 1000 / Details: highly purified, monodisperse preparation / Oligomeric state: hetero-hexamer / Number unique components: 6 |
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Molecular weight | Experimental: 420 KDa / Theoretical: 420 KDa / Method: Sedimentation |
-Macromolecule #1: Orc1p
Macromolecule | Name: 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 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 120 KDa / Theoretical: 120 KDa |
Recombinant expression | Organism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC1:6 |
Sequence | GO: nuclear origin of replication recognition complex / InterPro: AAA+ ATPase domain |
-Macromolecule #2: Orc2p
Macromolecule | Name: 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 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: yeast / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 73 KDa / Theoretical: 73 KDa |
Recombinant expression | Organism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC2:5 |
Sequence | GO: nuclear origin of replication recognition complex / InterPro: Origin recognition complex, subunit 2 |
-Macromolecule #3: Orc3p
Macromolecule | Name: Orc3p / type: protein_or_peptide / ID: 3 / Name.synonym: ORC subunit 3 / Details: third subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: yeast / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 72 KDa / Theoretical: 72 KDa |
Recombinant expression | Organism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC3:4 |
Sequence | GO: nuclear origin of replication recognition complex |
-Macromolecule #4: Orc4p
Macromolecule | Name: Orc4p / type: protein_or_peptide / ID: 4 / Name.synonym: ORC subunit 4 / Details: forth subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 61 KDa / Theoretical: 61 KDa |
Recombinant expression | Organism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC3:4 |
Sequence | GO: nuclear origin of replication recognition complex |
-Macromolecule #5: Orc5p
Macromolecule | Name: Orc5p / type: protein_or_peptide / ID: 5 / Name.synonym: ORC subunit 5 / Details: fifth subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 53 KDa / Theoretical: 53 KDa |
Recombinant expression | Organism: Hi-5 cell / Recombinant plasmid: baculoviruse bvORC2:5 |
Sequence | GO: nuclear origin of replication recognition complex |
-Macromolecule #6: Orc6p
Macromolecule | Name: Orc6p / type: protein_or_peptide / ID: 6 / Name.synonym: ORC subunit 6 / Details: smallest subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: yeast / synonym: baker's yeast / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus |
Molecular weight | Experimental: 50 KDa / Theoretical: 50 KDa |
Recombinant expression | Organism: Hi-5 Cell / Recombinant plasmid: baculovirus bvORC1:6 |
Sequence | GO: nuclear origin of replication recognition complex |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.02 mg/mL |
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Buffer | pH: 7.5 / Details: 50mM HEPES-KOH, 100mM KCl, 1mM EDTA, 1mM EGTA |
Staining | Type: NEGATIVE Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 30 seconds. |
Grid | Details: 300 mesh copper grid |
Vitrification | Cryogen name: NONE |
-Electron microscopy
Microscope | JEOL 1200EX |
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Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at |
Date | Oct 1, 2004 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 12.7 µm / Number real images: 60 / Average electron dose: 10 e/Å2 / Od range: 1.5 / Bits/pixel: 14 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated 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 stage | Specimen holder: Eucentric / Specimen holder model: OTHER / Tilt angle max: 45 |
-Image processing
CTF correction | Details: each film |
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Final reconstruction | Applied 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: 20000 |
Final two d classification | Number classes: 50 |