+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14439 | |||||||||||||||||||||||||||||||||
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Title | S. cerevisiae CMGE dimer nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
Map data | Consensus map of CMGE dimer model generated from EMD EMD-13978 | |||||||||||||||||||||||||||||||||
Sample |
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Keywords | DNA replication / helicase / initiation / DNA origin / REPLICATION | |||||||||||||||||||||||||||||||||
Function / homology | Function and homology information DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex ...DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex / DNA strand elongation involved in mitotic DNA replication / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / SUMO binding / Activation of the pre-replicative complex / CMG complex / single-stranded 3'-5' DNA helicase activity / nuclear pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / Termination of translesion DNA synthesis / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / single-stranded DNA helicase activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / mitotic sister chromatid cohesion / DNA replication proofreading / DNA strand elongation involved in DNA replication / leading strand elongation / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / Dual incision in TC-NER / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / DNA helicase activity / base-excision repair, gap-filling / helicase activity / replication fork / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA helicase / cell cycle / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) / DNA molecule (others) | |||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||||||||||||||||||||||||||
Authors | Lewis JS / Sousa JS | |||||||||||||||||||||||||||||||||
Funding support | European Union, France, United Kingdom, 10 items
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Citation | Journal: Nature / Year: 2022 Title: Mechanism of replication origin melting nucleated by CMG helicase assembly. Authors: Jacob S Lewis / Marta H Gross / Joana Sousa / Sarah S Henrikus / Julia F Greiwe / Andrea Nans / John F X Diffley / Alessandro Costa / Abstract: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex ...The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation. | |||||||||||||||||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14439.map.gz | 43.5 MB | EMDB map data format | |
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Header (meta data) | emd-14439-v30.xml emd-14439.xml | 40.2 KB 40.2 KB | Display Display | EMDB header |
Images | emd_14439.png | 83.3 KB | ||
Filedesc metadata | emd-14439.cif.gz | 13.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14439 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14439 | HTTPS FTP |
-Validation report
Summary document | emd_14439_validation.pdf.gz | 484.8 KB | Display | EMDB validaton report |
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Full document | emd_14439_full_validation.pdf.gz | 484.3 KB | Display | |
Data in XML | emd_14439_validation.xml.gz | 4.4 KB | Display | |
Data in CIF | emd_14439_validation.cif.gz | 4.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14439 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14439 | HTTPS FTP |
-Related structure data
Related structure data | 7z13MC 7qhsC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_14439.map.gz / Format: CCP4 / Size: 60.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Consensus map of CMGE dimer model generated from EMD EMD-13978 | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
+Entire : S. cerevisiae CMGE dimer nucleating origin DNA melting
+Supramolecule #1: S. cerevisiae CMGE dimer nucleating origin DNA melting
+Macromolecule #1: DNA replication licensing factor MCM2
+Macromolecule #2: DNA replication licensing factor MCM3
+Macromolecule #3: DNA replication licensing factor MCM4
+Macromolecule #4: DNA helicase
+Macromolecule #5: DNA replication licensing factor MCM6
+Macromolecule #6: DNA replication licensing factor MCM7
+Macromolecule #9: DNA replication complex GINS protein PSF3
+Macromolecule #10: DNA replication complex GINS protein SLD5
+Macromolecule #11: Cell division control protein 45
+Macromolecule #12: DNA polymerase epsilon subunit B
+Macromolecule #13: DNA replication complex GINS protein PSF1
+Macromolecule #14: DNA replication complex GINS protein PSF2
+Macromolecule #15: DNA polymerase epsilon catalytic subunit A
+Macromolecule #7: DNA (53-MER)
+Macromolecule #8: DNA (53-MER)
+Macromolecule #16: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #17: ZINC ION
+Macromolecule #18: MAGNESIUM ION
+Macromolecule #19: 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: 7.5 |
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
Details | four microlitres of sample was applied on a grid and incubated for 2 min at room temperature before blotting with filter paper for 5.5 s and plunge-freezing in liquid ethane. |
-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 / Number grids imaged: 2 / Number real images: 65286 / Average exposure time: 10.0 sec. / Average electron dose: 1.6 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.4 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 71348 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | One additional base pair has been built to connect the DNA molecules from the two individual symmetry expanded monomers. |
Refinement | Protocol: RIGID BODY FIT |
Output model | PDB-7z13: |