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Open data
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Basic information
Entry | Database: PDB / ID: 7z13 | |||||||||||||||||||||||||||||||||
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Title | S. cerevisiae CMGE dimer nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
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Function / homology | ![]() DNA-templated DNA replication maintenance of fidelity / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||||||||||||||||||||||||||
Method | ![]() ![]() ![]() | |||||||||||||||||||||||||||||||||
![]() | Lewis, J.S. / Sousa, J.S. / Costa, A. | |||||||||||||||||||||||||||||||||
Funding support | European Union, ![]() ![]()
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![]() | ![]() 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
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 2.4 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 14439MC ![]() 7qhsC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA replication licensing factor ... , 5 types, 10 molecules 2a3b4c6e7f
#1: Protein | Mass: 98911.539 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: PACBIOSEQ_LOCUS187, PACBIOSEQ_LOCUS193, PACBIOSEQ_LOCUS195, PACBIOSEQ_LOCUS196, SCNYR20_0007007400, SCP684_0007007100 Production host: ![]() ![]() ![]() ![]() #2: Protein | Mass: 111720.242 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: ![]() ![]() ![]() ![]() #3: Protein | Mass: 105138.375 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: ![]() ![]() ![]() ![]() #5: Protein | Mass: 113110.211 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: ![]() ![]() ![]() ![]() #6: Protein | Mass: 95049.875 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: MCM7, PACBIOSEQ_LOCUS429 / Production host: ![]() ![]() ![]() ![]() |
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-Protein , 2 types, 4 molecules 5dEL
#4: Protein | ![]() Mass: 86505.734 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: PACBIOSEQ_LOCUS4054, PACBIOSEQ_LOCUS4112, PACBIOSEQ_LOCUS4129, PACBIOSEQ_LOCUS4153, PACBIOSEQ_LOCUS4202, SCNYR20_0004029000, SCP684_0004028600 Production host: ![]() ![]() ![]() ![]() #11: Protein | Mass: 75154.703 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: ![]() ![]() ![]() |
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-DNA chain , 2 types, 2 molecules AB
#7: DNA chain | Mass: 16320.634 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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#8: DNA chain | Mass: 16311.620 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-DNA replication complex GINS protein ... , 4 types, 8 molecules CJDKHOIP
#9: Protein | Mass: 25718.070 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: PSF3, YOL146W / Production host: ![]() ![]() ![]() #10: Protein | Mass: 33983.617 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: SLD5, YDR489W / Production host: ![]() ![]() ![]() #13: Protein | Mass: 24230.576 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: PACBIOSEQ_LOCUS944, PACBIOSEQ_LOCUS956, PACBIOSEQ_LOCUS958, SCNYR20_0001022500, SCP684_0001022000 Production host: ![]() ![]() ![]() #14: Protein | Mass: 25096.807 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: PACBIOSEQ_LOCUS3163, PACBIOSEQ_LOCUS3191, PACBIOSEQ_LOCUS3224, PACBIOSEQ_LOCUS3231, PACBIOSEQ_LOCUS3255, SCNYR20_0009012300, SCP684_0009011800 Production host: ![]() ![]() ![]() |
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-DNA polymerase epsilon ... , 2 types, 4 molecules FMNQ
#12: Protein | ![]() Mass: 78425.852 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: DPB2, YPR175W, P9705.7 / Production host: ![]() ![]() ![]() #15: Protein | Mass: 255992.484 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Strain: ATCC 204508 / S288c / Gene: POL2, DUN2, YNL262W, N0825 / Production host: ![]() ![]() ![]() References: UniProt: P21951, ![]() ![]() |
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-Non-polymers , 4 types, 32 molecules ![](data/chem/img/ATP.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/ADP.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/ADP.gif)
#16: Chemical | ChemComp-ATP / ![]() #17: Chemical | ChemComp-ZN / #18: Chemical | ChemComp-MG / #19: Chemical | ChemComp-ADP / ![]() |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: S. cerevisiae CMGE dimer nucleating origin DNA melting Type: COMPLEX Details: Dimeric model reconstituted from symmetry expanded monomer (PDB entry 7QHS). Entity ID: #1-#15 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() 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. |
Vitrification![]() | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 1.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 65286 |
Image scans | Movie frames/image: 32 |
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Processing
EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71348 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT Details: One additional base pair has been built to connect the DNA molecules from the two individual symmetry expanded monomers. | ||||||||||||||||||||||||
Atomic model building |
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