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Yorodumi- EMDB-21665: Cryo-EM map of pre-insertion mutant OCCM-DNA complex (ORC-Cdc6-Cd... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-21665 | ||||||||||||||||||
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Title | Cryo-EM map of pre-insertion mutant OCCM-DNA complex (ORC-Cdc6-Cdt1-Mcm2-7 with Mcm6 WHD truncation) | ||||||||||||||||||
Map data | |||||||||||||||||||
Sample |
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Keywords | DNA replication / Cryo-EM / OCCM-deltaC6 / REPLICATION-DNA complex | ||||||||||||||||||
Function / homology | Function and homology information MCM complex loading / CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / Assembly of the ORC complex at the origin of replication ...MCM complex loading / CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / Assembly of the ORC complex at the origin of replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nuclear origin of replication recognition complex / mitotic DNA replication / Activation of the pre-replicative complex / nucleosome organization / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / cyclin-dependent protein serine/threonine kinase inhibitor activity / mitotic DNA replication checkpoint signaling / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / CDK-mediated phosphorylation and removal of Cdc6 / DNA unwinding involved in DNA replication / Orc1 removal from chromatin / nuclear replication fork / regulation of DNA replication / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / nucleosome binding / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / G1/S transition of mitotic cell cycle / single-stranded DNA binding / chromosome / DNA helicase / chromosome, telomeric region / cell division / GTPase activity / DNA damage response / chromatin binding / GTP binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | ||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.1 Å | ||||||||||||||||||
Authors | Yuan Z / Schneider S | ||||||||||||||||||
Funding support | United States, United Kingdom, 5 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2020 Title: Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6. Authors: Zuanning Yuan / Sarah Schneider / Thomas Dodd / Alberto Riera / Lin Bai / Chunli Yan / Indiana Magdalou / Ivaylo Ivanov / Bruce Stillman / Huilin Li / Christian Speck / Abstract: DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M- ...DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication. | ||||||||||||||||||
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_21665.map.gz | 59.4 MB | EMDB map data format | |
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Header (meta data) | emd-21665-v30.xml emd-21665.xml | 37.2 KB 37.2 KB | Display Display | EMDB header |
Images | emd_21665.png | 148.2 KB | ||
Filedesc metadata | emd-21665.cif.gz | 12.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-21665 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-21665 | HTTPS FTP |
-Validation report
Summary document | emd_21665_validation.pdf.gz | 489.2 KB | Display | EMDB validaton report |
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Full document | emd_21665_full_validation.pdf.gz | 488.7 KB | Display | |
Data in XML | emd_21665_validation.xml.gz | 6.1 KB | Display | |
Data in CIF | emd_21665_validation.cif.gz | 6.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-21665 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-21665 | HTTPS FTP |
-Related structure data
Related structure data | 6wggMC 6wgcC 6wgfC 6wgiC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_21665.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.31 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 : ORC-Cdc6-Mcm2-7-dsDNA
+Supramolecule #1: ORC-Cdc6-Mcm2-7-dsDNA
+Macromolecule #1: Cell division cycle protein CDT1
+Macromolecule #2: Cell division control protein 6
+Macromolecule #3: Origin recognition complex subunit 1
+Macromolecule #4: Origin recognition complex subunit 2
+Macromolecule #5: Origin recognition complex subunit 3
+Macromolecule #6: Origin recognition complex subunit 5
+Macromolecule #7: Origin recognition complex subunit 4
+Macromolecule #8: Origin recognition complex subunit 6
+Macromolecule #11: DNA replication licensing factor MCM2
+Macromolecule #12: DNA replication licensing factor MCM3
+Macromolecule #13: DNA replication licensing factor MCM4
+Macromolecule #14: Minichromosome maintenance protein 5
+Macromolecule #15: DNA replication licensing factor MCM6
+Macromolecule #16: DNA replication licensing factor MCM7
+Macromolecule #9: DNA (41-MER)
+Macromolecule #10: DNA (41-MER)
+Macromolecule #17: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 25126 |
Initial angle assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: PROJECTION MATCHING |
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | PDB-6wgg: |