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- PDB-6skl: Cryo-EM structure of the CMG Fork Protection Complex at a replica... -
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Basic information
Entry | Database: PDB / ID: 6skl | ||||||
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Title | Cryo-EM structure of the CMG Fork Protection Complex at a replication fork - Conformation 1 | ||||||
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![]() | REPLICATION / protein-DNA complex / replisome / AAA+ helicase / CMG / GINS / fork DNA / MCM / fork protection complex / CIP-box | ||||||
Function / homology | ![]() establishment of sister chromatid cohesion / maintenance of DNA repeat elements / Unwinding of DNA / replication fork arrest / Cul8-RING ubiquitin ligase complex / meiotic chromosome segregation / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding ...establishment of sister chromatid cohesion / maintenance of DNA repeat elements / Unwinding of DNA / replication fork arrest / Cul8-RING ubiquitin ligase complex / meiotic chromosome segregation / 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 / Activation of the pre-replicative complex / CMG complex / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / single-stranded 3'-5' DNA helicase activity / entrainment of circadian clock / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / replication fork protection complex / mitotic DNA replication initiation / double-strand break repair via break-induced replication / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / single-stranded DNA helicase activity / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / nuclear chromosome / replication fork processing / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / heterochromatin formation / helicase activity / meiotic cell cycle / DNA-templated DNA replication / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / DNA replication / DNA helicase / chromosome, telomeric region / DNA repair / DNA damage response / chromatin binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å | ||||||
Model details | CMG-Csm3-Tof1-Mrc1-Ctf4-DNA | ||||||
![]() | Yeeles, J. / Baretic, D. / Jenkyn-Bedford, M. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork. Authors: Domagoj Baretić / Michael Jenkyn-Bedford / Valentina Aria / Giuseppe Cannone / Mark Skehel / Joseph T P Yeeles / ![]() Abstract: The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric ...The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability. | ||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 2.5 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 1.5 MB | Display | |
Data in XML | ![]() | 174.8 KB | Display | |
Data in CIF | ![]() | 280.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 10227MC ![]() 6skoC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA replication licensing factor ... , 5 types, 5 molecules 23467
#1: Protein | Mass: 98911.539 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM2, YBL023C, YBL0438 / Production host: ![]() ![]() |
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#2: Protein | Mass: 107653.508 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: ![]() ![]() |
#3: Protein | Mass: 105138.375 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: ![]() ![]() |
#5: Protein | Mass: 113110.211 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM6, YGL201C / Production host: ![]() ![]() |
#6: Protein | Mass: 95049.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: ![]() ![]() |
-Protein , 5 types, 7 molecules 5EFGHXY
#4: Protein | Mass: 86505.734 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: ![]() ![]() | ||||
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#11: Protein | Mass: 74324.836 Da / Num. of mol.: 1 / Fragment: Mcm4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: ![]() ![]() | ||||
#12: Protein | Mass: 104543.391 Da / Num. of mol.: 3 / Fragment: Mcm6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: CTF4, CHL15, POB1, YPR135W, P9659.7 / Production host: ![]() ![]() #15: Protein | | Mass: 141296.875 Da / Num. of mol.: 1 / Fragment: Mcm2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: TOF1, YNL273W, N0636 / Production host: ![]() ![]() #16: Protein | | Mass: 36402.590 Da / Num. of mol.: 1 / Fragment: Mcm3 / Mutation: CBP-tag at N-terminus Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: CSM3, YMR048W, YM9796.01 / Production host: ![]() ![]() |
-DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD
#7: Protein | Mass: 24230.576 Da / Num. of mol.: 1 / Fragment: Tof1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: ![]() ![]() |
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#8: Protein | Mass: 25096.807 Da / Num. of mol.: 1 / Fragment: Csm3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PSF2, YJL072C, HRF213, J1086 / Production host: ![]() ![]() |
#9: Protein | Mass: 24437.859 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: His-tag at the N-terminus (24 residues) Source: (gene. exp.) ![]() ![]() Gene: PSF3, YOL146W / Production host: ![]() ![]() |
#10: Protein | Mass: 33983.617 Da / Num. of mol.: 1 / Fragment: Ctf4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: SLD5, YDR489W / Production host: ![]() ![]() |
-DNA chain , 2 types, 2 molecules IJ
#13: DNA chain | Mass: 26396.836 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: Cy3-label at 5'-end. Five phosphorothioate backbone linkages between residues at the very 3'-end. The dT 16 residues from the 5'-end was biotinylated. Source: (synth.) synthetic construct (others) |
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#14: DNA chain | Mass: 18524.887 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: dT (residue 47) biotinylated. / Source: (synth.) synthetic construct (others) |
-Non-polymers , 3 types, 11 molecules ![](data/chem/img/ZN.gif)
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![](data/chem/img/MG.gif)
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#17: Chemical | ChemComp-ZN / #18: Chemical | #19: Chemical | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 1.4 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: In vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork | |||||||||||||||||||||||||||||||||||
Specimen support | Details: 15 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277.15 K Details: Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid 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 / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 7 sec. / Electron dose: 37 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6682 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 20 / Used frames/image: 1-20 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 632000 | ||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 20 / Protocol: OTHER / Space: REAL / Target criteria: FSC 0.5 | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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