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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4980 | |||||||||
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Title | Cryo-EM structure of an MCM loading intermediate | |||||||||
![]() | Full map - complete MCM-ORC (MO) origin licensing intermediate | |||||||||
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![]() | DNA Replication / Origin licensing / MCM2-7 helicase / Origin Recognition Complex / REPLICATION | |||||||||
Function / homology | ![]() 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 / nuclear origin of replication recognition complex ...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 / nuclear origin of replication recognition complex / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nucleosome organization / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / 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 / 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 / single-stranded DNA binding / chromosome, telomeric region / DNA helicase / chromatin binding / DNA damage response / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.4 Å | |||||||||
![]() | Miller TCR / Locke J | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Authors: Thomas C R Miller / Julia Locke / Julia F Greiwe / John F X Diffley / Alessandro Costa / ![]() Abstract: In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double- ...In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 9.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 62.1 KB 62.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() ![]() ![]() | 12.1 KB 12.1 KB 12.1 KB | Display Display Display | ![]() |
Images | ![]() | 131.5 KB | ||
Masks | ![]() ![]() ![]() | 149.9 MB 149.9 MB 149.9 MB | ![]() | |
Filedesc metadata | ![]() | 13.7 KB | ||
Others | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | 96.8 MB 96.8 MB 8.4 MB 108.5 MB 6.5 MB 108.5 MB 118.1 MB 118.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6rqcMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Full map - complete MCM-ORC (MO) origin licensing intermediate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.38 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
+Mask #1
+Mask #2
+Mask #3
+Additional map: Half map - MCM-Orc6N lobe of MCM-ORC (MO)...
+Additional map: Half map - MCM-Orc6N lobe of MCM-ORC (MO)...
+Additional map: Full map - MCM-Orc6N lobe of MCM-ORC (MO)...
+Additional map: Half map - Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+Additional map: Full map - Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+Additional map: Half map – Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+Half map: Half map - complete MCM-ORC (MO) origin licensing intermediate
+Half map: Half map - complete MCM-ORC (MO) origin licensing intermediate
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Sample components
+Entire : The MCM-ORC (MO) loading intermediate
+Supramolecule #1: The MCM-ORC (MO) loading intermediate
+Supramolecule #2: MCM-Orc6N lobe of the MCM-ORC (MO) origin licensing intermediate.
+Supramolecule #3: Orc1-5-Orc6C lobe of the MCM-ORC (MO) origin licensing intermediate
+Supramolecule #4: The MCM-ORC (MO) loading intermediate protein complex
+Supramolecule #5: DNA
+Macromolecule #1: Origin recognition complex subunit 1
+Macromolecule #2: Origin recognition complex subunit 2
+Macromolecule #3: Origin recognition complex subunit 3
+Macromolecule #4: Origin recognition complex subunit 4
+Macromolecule #5: Origin recognition complex subunit 5
+Macromolecule #6: Origin recognition complex subunit 6
+Macromolecule #7: DNA replication licensing factor MCM2
+Macromolecule #8: DNA replication licensing factor MCM3
+Macromolecule #9: DNA replication licensing factor MCM4
+Macromolecule #10: Minichromosome maintenance protein 5
+Macromolecule #11: DNA replication licensing factor MCM6
+Macromolecule #12: DNA replication licensing factor MCM7
+Macromolecule #13: DNA (88-MER)
+Macromolecule #14: DNA (88-MER)
+Macromolecule #15: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #16: MAGNESIUM ION
+Macromolecule #17: ADENOSINE-5'-DIPHOSPHATE
+Macromolecule #18: ZINC ION
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.6 Component:
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Grid | Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 288 K / Instrument: LEICA EM GP Details: 10 second incubation, 3.5 seconds single side blotting.. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Average electron dose: 1.68 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||||||
Output model | ![]() PDB-6rqc: |