+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13176 | |||||||||||||||
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Title | 3.0 A resolution structure of a DNA-loaded MCM double hexamer | |||||||||||||||
Map data | ||||||||||||||||
Sample |
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Keywords | Mcm2-7 helicase / nucleoprotein complex / AAA+ ATPase / DNA replication / REPLICATION | |||||||||||||||
Function / homology | Function and homology information MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex ...MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication initiation / 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 / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Saccharomyces cerevisiae S288C (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||||||||
Authors | Greiwe JF / Locke J / Nans A / Costa A | |||||||||||||||
Funding support | European Union, 4 items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Authors: Julia F Greiwe / Thomas C R Miller / Julia Locke / Fabrizio Martino / Steven Howell / Anne Schreiber / Andrea Nans / John F X Diffley / Alessandro Costa / Abstract: Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM ...Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional 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_13176.map.gz | 59.6 MB | EMDB map data format | |
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Header (meta data) | emd-13176-v30.xml emd-13176.xml | 35.9 KB 35.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13176_fsc.xml | 16.9 KB | Display | FSC data file |
Images | emd_13176.png | 149.1 KB | ||
Filedesc metadata | emd-13176.cif.gz | 10.4 KB | ||
Others | emd_13176_additional_1.map.gz emd_13176_additional_2.map.gz emd_13176_half_map_1.map.gz emd_13176_half_map_2.map.gz | 393.5 MB 335.8 MB 335.5 MB 335.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13176 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13176 | HTTPS FTP |
-Validation report
Summary document | emd_13176_validation.pdf.gz | 956.8 KB | Display | EMDB validaton report |
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Full document | emd_13176_full_validation.pdf.gz | 956.4 KB | Display | |
Data in XML | emd_13176_validation.xml.gz | 23.8 KB | Display | |
Data in CIF | emd_13176_validation.cif.gz | 30.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13176 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13176 | HTTPS FTP |
-Related structure data
Related structure data | 7p30MC 7p5zC M: atomic model generated by this map C: citing same article (ref.) |
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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_13176.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: 1.08 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: #1
File | emd_13176_additional_1.map | ||||||||||||
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Density Histograms |
-Additional map: #2
File | emd_13176_additional_2.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_13176_half_map_1.map | ||||||||||||
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Density Histograms |
-Half map: #1
File | emd_13176_half_map_2.map | ||||||||||||
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Density Histograms |
-Sample components
+Entire : S. cerevisiae MCM double hexamer bound to duplex DNA
+Supramolecule #1: S. cerevisiae MCM double hexamer bound to duplex DNA
+Macromolecule #1: DNA replication licensing factor MCM2
+Macromolecule #2: DNA replication licensing factor MCM3
+Macromolecule #3: DNA replication licensing factor MCM4
+Macromolecule #4: Minichromosome maintenance protein 5
+Macromolecule #5: DNA replication licensing factor MCM6
+Macromolecule #6: DNA replication licensing factor MCM7
+Macromolecule #7: DNA (53-MER)
+Macromolecule #8: DNA (53-MER)
+Macromolecule #9: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #10: MAGNESIUM ION
+Macromolecule #11: ZINC ION
+Macromolecule #12: ADENOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3 seconds before plunging. |
Details | The entire MCM loading and phosphorylation reaction was applied to the EM grid. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 18123 / Average exposure time: 9.0 sec. / Average electron dose: 51.3 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.1 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Protocol: FLEXIBLE FIT | ||||||||
Output model | PDB-7p30: |