|Entry||Database: PDB / ID: 6n7i|
|Title||Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (gp4(5)-DNA)|
|Keywords||HYDROLASE / TRANSFERASE/DNA / helicase / ATPase / hexamer / DNA replication / TRANSFERASE-DNA complex|
|Function / homology||TOPRIM domain / DNA helicase, DnaB-like, C-terminal / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Archaeal primase DnaG/twinkle, TOPRIM domain / DnaB-like helicase C terminal domain / Zinc-binding domain of primase-helicase / Toprim domain profile. / Superfamily 4 helicase domain profile. / DNA primase activity ...TOPRIM domain / DNA helicase, DnaB-like, C-terminal / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Archaeal primase DnaG/twinkle, TOPRIM domain / DnaB-like helicase C terminal domain / Zinc-binding domain of primase-helicase / Toprim domain profile. / Superfamily 4 helicase domain profile. / DNA primase activity / DNA helicase activity / DNA helicase / Transferases, Transferring phosphorus-containing groups, Nucleotidyltransferases / zinc ion binding / ATP binding / DNA primase/helicase|
Function and homology information
|Specimen source||Enterobacteria phage T7 (bacteriophage)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution|
|Authors||Gao, Y. / Cui, Y. / Zhou, Z. / Yang, W.|
|Citation||Journal: Science / Year: 2019|
Title: Structures and operating principles of the replisome.
Authors: Yang Gao / Yanxiang Cui / Tara Fox / Shiqiang Lin / Huaibin Wang / Natalia de Val / Z Hong Zhou / Wei Yang
Abstract: Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model ...Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.
SummaryFull reportAbout validation report
|Date||Deposition: Nov 27, 2018 / Release: Mar 6, 2019|
|Structure viewer||Molecule: |
Downloads & links
A: DNA primase/helicase
B: DNA primase/helicase
C: DNA primase/helicase
D: DNA primase/helicase
E: DNA primase/helicase
F: DNA primase/helicase
T: DNA (25-MER)
Mass: 62734.430 Da / Num. of mol.: 6 / Mutation: E343Q
Source: (gene. exp.) Enterobacteria phage T7 (bacteriophage)
Variant: E343Q / Production host: Escherichia coli (E. coli)
References: UniProt: P03692, Transferases, Transferring phosphorus-containing groups, Nucleotidyltransferases, DNA helicase
|#2: DNA chain|
Mass: 7594.877 Da / Num. of mol.: 1 / Source: (synth.) Enterobacteria phage T7 (bacteriophage)
|#5: Water|| ChemComp-HOH / |
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex with five helicase subunits ordered|
Type: COMPLEX / Entity ID: 1,
|Source (natural)||Organism: Enterobacteria phage T7 (bacteriophage)||Source (recombinant)||Organism: Escherichia coli (E. coli)||Buffer solution||pH: 7.5||Buffer component|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Specimen support||Grid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R1.2/1.3||Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 kelvins|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C1|
|3D reconstruction||Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 227594 / Symmetry type: POINT|
|Atomic model building||Ref protocol: FLEXIBLE FIT / Ref space: REAL|
|Atomic model building||PDB-ID: 1E0J|
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