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Yorodumi- 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 | ||||||
Components |
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Keywords | REPLICATION / protein-DNA complex / replisome / AAA+ helicase / CMG / GINS / fork DNA / MCM / fork protection complex / CIP-box | ||||||
Function / homology | Function and homology information 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 / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / nuclear chromosome / DNA unwinding involved in DNA replication / replication fork processing / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / meiotic cell cycle / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / chromosome, telomeric region / DNA replication / DNA helicase / DNA repair / chromatin binding / DNA damage response / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å | ||||||
Model details | CMG-Csm3-Tof1-Mrc1-Ctf4-DNA | ||||||
Authors | Yeeles, J. / Baretic, D. / Jenkyn-Bedford, M. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Mol Cell / Year: 2020 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
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6skl.cif.gz | 2.5 MB | Display | PDBx/mmCIF format |
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PDB format | pdb6skl.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 6skl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6skl_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 6skl_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 6skl_validation.xml.gz | 174.8 KB | Display | |
Data in CIF | 6skl_validation.cif.gz | 280.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sk/6skl ftp://data.pdbj.org/pub/pdb/validation_reports/sk/6skl | HTTPS FTP |
-Related structure data
Related structure data | 10227MC 6skoC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, DNA helicase |
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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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase |
#3: Protein | Mass: 105138.375 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase |
#5: Protein | Mass: 113110.211 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase |
#6: Protein | Mass: 95049.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase |
-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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, DNA helicase | ||||
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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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032 | ||||
#12: Protein | Mass: 104543.391 Da / Num. of mol.: 3 / Fragment: Mcm6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: CTF4, CHL15, POB1, YPR135W, P9659.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q01454 #15: Protein | | Mass: 141296.875 Da / Num. of mol.: 1 / Fragment: Mcm2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: TOF1, YNL273W, N0636 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53840 #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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: CSM3, YMR048W, YM9796.01 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04659 |
-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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488 |
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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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359 |
#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.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: PSF3, YOL146W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12146 |
#10: Protein | Mass: 33983.617 Da / Num. of mol.: 1 / Fragment: Ctf4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406 |
-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
#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 |
-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. |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
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 |
-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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