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- PDB-5u8t: Structure of Eukaryotic CMG Helicase at a Replication Fork and Im... -
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Basic information
Entry | Database: PDB / ID: 5u8t | |||||||||
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Title | Structure of Eukaryotic CMG Helicase at a Replication Fork and Implications | |||||||||
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![]() | REPLICATION / CMG helicase / replisome / origin initiation / DNA polymerase / DNA replication | |||||||||
Function / homology | ![]() Unwinding of DNA / 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 ...Unwinding of DNA / 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 / 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 / DNA strand elongation involved in DNA replication / 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 | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å | |||||||||
![]() | Li, B. / Georgescu, R. / Yuan, Z. / Santos, R. / Sun, J. / Zhang, D. / Yurieva, O. / Li, H. / O'Donnell, M.E. | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1 MB | Display | ![]() |
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PDB format | ![]() | 850.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 8519MC ![]() 8518C ![]() 8520C ![]() 5u8sC 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.) ![]() ![]() Strain: ATCC 204508 / S288c / 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.) ![]() ![]() Strain: ATCC 204508 / S288c / 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.) ![]() ![]() Strain: ATCC 204508 / S288c / 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.) ![]() ![]() Strain: ATCC 204508 / S288c / 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.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: ![]() ![]() |
-Protein , 2 types, 2 molecules 5E
#4: Protein | Mass: 86505.734 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: ![]() ![]() |
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#11: Protein | Mass: 74324.836 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: ![]() ![]() |
-DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD
#7: Protein | Mass: 24230.576 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: ![]() ![]() |
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#8: Protein | Mass: 25096.807 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: PSF2, YJL072C, HRF213, J1086 / Production host: ![]() ![]() |
#9: Protein | Mass: 21977.135 Da / Num. of mol.: 1 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.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 204508 / S288c / Gene: SLD5, YDR489W / Production host: ![]() ![]() |
-DNA chain / Non-polymers , 2 types, 4 molecules F![](data/chem/img/ANP.gif)
![](data/chem/img/ANP.gif)
#12: DNA chain | Mass: 4213.742 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Production host: ![]() ![]() |
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#13: Chemical |
-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
Component | Name: CMG-ssDNA / Type: COMPLEX / Entity ID: #1-#12 / Source: RECOMBINANT |
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Molecular weight | Value: 0.75 MDa |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | 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 |
Image recording | Electron dose: 10 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.9_1692 / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 395443 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 4.9→332.8 Å / SU ML: 1.45 / Phase error: 54.63 / Stereochemistry target values: MLHL
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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LS refinement shell |
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