[English] 日本語
Yorodumi- EMDB-10227: Cryo-EM structure of the CMG Fork Protection Complex at a replica... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10227 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of the CMG Fork Protection Complex at a replication fork - Conformation 1 | |||||||||
Map data | Conformation1 CMG-Csm3-Tof1-Mrc1-Ctf4 and a DNA fork | |||||||||
Sample |
| |||||||||
Keywords | protein-DNA complex / replisome / AAA+ helicase / CMG / GINS / fork DNA / MCM / fork protection complex / CIP-box / REPLICATION | |||||||||
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 / DNA strand elongation involved in mitotic DNA replication ...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 / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / GINS complex / 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 / nuclear pre-replicative complex / DNA replication checkpoint signaling / establishment of mitotic sister chromatid cohesion / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / nuclear chromosome / DNA unwinding involved in DNA replication / replication fork processing / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / DNA helicase activity / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / DNA helicase / DNA replication / 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 / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
Authors | Baretic D / Jenkyn-Bedford M | |||||||||
Funding support | United Kingdom, 1 items
| |||||||||
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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_10227.map.gz | 161.5 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-10227-v30.xml emd-10227.xml | 46.5 KB 46.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10227_fsc.xml | 12.8 KB | Display | FSC data file |
Images | emd_10227.png | 106.5 KB | ||
Filedesc metadata | emd-10227.cif.gz | 14.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10227 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10227 | HTTPS FTP |
-Validation report
Summary document | emd_10227_validation.pdf.gz | 650.7 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_10227_full_validation.pdf.gz | 650.3 KB | Display | |
Data in XML | emd_10227_validation.xml.gz | 13 KB | Display | |
Data in CIF | emd_10227_validation.cif.gz | 17.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10227 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10227 | HTTPS FTP |
-Related structure data
Related structure data | 6sklMC 6skoC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_10227.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Conformation1 CMG-Csm3-Tof1-Mrc1-Ctf4 and a DNA fork | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.049 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
+Entire : Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
+Supramolecule #1: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
+Supramolecule #2: CMG-Csm3-Tof1-Mrc1-Ctf4
+Supramolecule #3: DNA
+Macromolecule #1: DNA replication licensing factor MCM2
+Macromolecule #2: DNA replication licensing factor MCM3
+Macromolecule #3: DNA replication licensing factor MCM4
+Macromolecule #4: Minichromosome maintenance protein 5
+Macromolecule #5: DNA replication licensing factor MCM6
+Macromolecule #6: DNA replication licensing factor MCM7
+Macromolecule #7: DNA replication complex GINS protein PSF1
+Macromolecule #8: DNA replication complex GINS protein PSF2
+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 alpha-binding protein
+Macromolecule #15: Topoisomerase 1-associated factor 1
+Macromolecule #16: Chromosome segregation in meiosis protein 3
+Macromolecule #13: DNA fork, leading-strand template
+Macromolecule #14: DNA fork, lagging-strand template
+Macromolecule #17: ZINC ION
+Macromolecule #18: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
+Macromolecule #19: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| |||||||||||||||||||||
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER 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.. | |||||||||||||||||||||
Details | In vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 2 / Number real images: 6682 / Average exposure time: 7.0 sec. / Average electron dose: 37.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 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
-Atomic model buiding 1
Initial model |
| ||||||||
---|---|---|---|---|---|---|---|---|---|
Refinement | Space: REAL / Protocol: OTHER / Overall B value: 20 / Target criteria: FSC 0.5 | ||||||||
Output model | PDB-6skl: |