+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8518 | |||||||||
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Title | Structure of eukaryotic CMG helicase at a replication fork | |||||||||
Map data | Yeast CMG with biotinylated fork DNA and streptavidin | |||||||||
Sample |
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Keywords | CMG helicase / replisome / origin initiation / DNA polymerase / DNA replication / REPLICATION | |||||||||
Function / homology | Function and homology information Unwinding of DNA / 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 ...Unwinding of DNA / 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 / 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 / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.1 Å | |||||||||
Authors | Li H / Li B | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2017 Title: Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation. Authors: Roxana Georgescu / Zuanning Yuan / Lin Bai / Ruda de Luna Almeida Santos / Jingchuan Sun / Dan Zhang / Olga Yurieva / Huilin Li / Michael E O'Donnell / Abstract: The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ...The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8518.map.gz | 59.8 MB | EMDB map data format | |
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Header (meta data) | emd-8518-v30.xml emd-8518.xml | 30.9 KB 30.9 KB | Display Display | EMDB header |
Images | emd_8518.png | 98.8 KB | ||
Filedesc metadata | emd-8518.cif.gz | 10.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8518 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8518 | HTTPS FTP |
-Validation report
Summary document | emd_8518_validation.pdf.gz | 519.9 KB | Display | EMDB validaton report |
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Full document | emd_8518_full_validation.pdf.gz | 519.4 KB | Display | |
Data in XML | emd_8518_validation.xml.gz | 6.2 KB | Display | |
Data in CIF | emd_8518_validation.cif.gz | 7.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8518 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8518 | HTTPS FTP |
-Related structure data
Related structure data | 5u8sMC 8519C 8520C 5u8tC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_8518.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Yeast CMG with biotinylated fork DNA and streptavidin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : CMG-biotinylated fork DNA-Streptavidin
+Supramolecule #1: CMG-biotinylated fork DNA-Streptavidin
+Macromolecule #1: DNA replication complex GINS protein PSF1
+Macromolecule #2: DNA replication complex GINS protein PSF2
+Macromolecule #3: DNA replication complex GINS protein PSF3
+Macromolecule #4: DNA replication complex GINS protein SLD5
+Macromolecule #5: Cell division control protein 45
+Macromolecule #8: DNA replication licensing factor MCM2
+Macromolecule #9: DNA replication licensing factor MCM3
+Macromolecule #10: DNA replication licensing factor MCM4
+Macromolecule #11: Minichromosome maintenance protein 5
+Macromolecule #12: DNA replication licensing factor MCM6
+Macromolecule #13: DNA replication licensing factor MCM7
+Macromolecule #6: DNA (26-MER)
+Macromolecule #7: DNA (5'-D(P*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')
+Macromolecule #14: ADENOSINE-5'-TRIPHOSPHATE
-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 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 10.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: OTHER / Details: EMD-6535 |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 243796 |
Initial angle assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: PROJECTION MATCHING |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
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Output model | PDB-5u8s: |