+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13978 | |||||||||||||||||||||||||||||||||
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Title | S. cerevisiae CMGE nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
Map data | PEHNIX Resolve cryoEM density modified map generated from RELION half maps | |||||||||||||||||||||||||||||||||
Sample |
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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 / nuclear DNA replication / MCM complex binding ...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 / nuclear DNA replication / MCM complex binding / GINS complex / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nucleotide-excision repair, DNA gap filling / SUMO binding / mitotic DNA replication / DNA replication proofreading / Activation of the pre-replicative complex / CMG complex / single-stranded DNA 3'-5' DNA exonuclease activity / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication checkpoint signaling / replication fork protection complex / mitotic DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA strand elongation involved in DNA replication / mitotic sister chromatid cohesion / leading strand elongation / nuclear chromosome / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / base-excision repair, gap-filling / DNA helicase activity / replication fork / helicase activity / protein-DNA complex / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA helicase / DNA replication / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / cell cycle / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) / DNA molecule (others) | |||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||||||||||||||||||||
Authors | Lewis JS / Sousa JS / Costa A | |||||||||||||||||||||||||||||||||
Funding support | European Union, 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_13978.map.gz | 23.5 MB | EMDB map data format | |
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Header (meta data) | emd-13978-v30.xml emd-13978.xml | 46.6 KB 46.6 KB | Display Display | EMDB header |
Images | emd_13978.png | 48.7 KB | ||
Others | emd_13978_additional_1.map.gz emd_13978_half_map_1.map.gz emd_13978_half_map_2.map.gz | 478.5 MB 410.8 MB 411 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13978 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13978 | HTTPS FTP |
-Related structure data
Related structure data | 7qhsMC 7z13C C: citing same article (ref.) M: atomic model generated by this map |
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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_13978.map.gz / Format: CCP4 / Size: 25.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | PEHNIX Resolve cryoEM density modified map generated from RELION half maps | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||
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
-Additional map: RELION 3.1 postprocessed map generated from RELION half maps
File | emd_13978_additional_1.map | ||||||||||||
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Annotation | RELION 3.1 postprocessed map generated from RELION half maps | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: RELION half map 1
File | emd_13978_half_map_1.map | ||||||||||||
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Annotation | RELION half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: RELION half map 2
File | emd_13978_half_map_2.map | ||||||||||||
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Annotation | RELION half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : S. cerevisiae CMG helicase nucleating origin DNA melting
+Supramolecule #1: S. cerevisiae CMG helicase 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 replication licensing factor MCM6
+Macromolecule #5: DNA replication licensing factor MCM7
+Macromolecule #6: DNA replication complex GINS protein PSF1
+Macromolecule #7: DNA replication complex GINS protein PSF2
+Macromolecule #8: DNA replication complex GINS protein PSF3
+Macromolecule #9: DNA replication complex GINS protein SLD5
+Macromolecule #10: Cell division control protein 45
+Macromolecule #11: DNA polymerase epsilon subunit B
+Macromolecule #12: DNA polymerase epsilon catalytic subunit A
+Macromolecule #15: DNA replication licensing factor MCM5
+Macromolecule #13: DNA (26-MER)
+Macromolecule #14: DNA (26-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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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 FIELDBright-field microscopy / 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 |
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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 927109 |
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CTF correction | Software: (Name: RELION (ver. 3.1), Gctf (ver. 1.18)) |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) Details: Reported resolution after PHENIX resolve cryoEM density modification. Input were half maps generated in RELION 3.1 Number images used: 71348 |