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- PDB-6n9v: Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: PDB / ID: 6n9v
TitleStructure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS1)
Components
  • DNA primase/helicase
  • DNA-directed DNA polymeraseDNA polymerase
  • Primer
  • Template
KeywordsHYDROLASE / TRANSFERASE/DNA / DNA polymerase / primase / helicase / DNA replication / replisome / TRANSFERASE-DNA complex
Function / homologyTOPRIM domain / DNA-directed DNA polymerase T7 / P-loop containing nucleoside triphosphate hydrolase / DNA-directed DNA polymerase, family A, conserved site / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Ribonuclease H-like superfamily / DNA helicase, DnaB-like, C-terminal / Ribonuclease H superfamily / DNA polymerase A / DNA-directed DNA polymerase, family A, palm domain ...TOPRIM domain / DNA-directed DNA polymerase T7 / P-loop containing nucleoside triphosphate hydrolase / DNA-directed DNA polymerase, family A, conserved site / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Ribonuclease H-like superfamily / DNA helicase, DnaB-like, C-terminal / Ribonuclease H superfamily / DNA polymerase A / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase family A / Archaeal primase DnaG/twinkle, TOPRIM domain / DnaB-like helicase C terminal domain / Zinc-binding domain of primase-helicase / DNA polymerase family A signature. / Toprim domain profile. / Superfamily 4 helicase domain profile. / DNA primase activity / viral DNA genome replication / DNA helicase activity / Hydrolases, Acting on ester bonds, Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA helicase / 3'-5' exonuclease activity / DNA-dependent DNA replication / Transferases, Transferring phosphorus-containing groups, Nucleotidyltransferases / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / zinc ion binding / ATP binding / metal ion binding / DNA-directed DNA polymerase / DNA primase/helicase
Function and homology information
Specimen sourceEnterobacteria phage T7 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4 Å resolution
AuthorsGao, Y. / Fox, T. / Val, N. / Yang, W.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute1S10RR23057United States
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 4, 2018 / Release: Mar 6, 2019

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Structure visualization

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Assembly

Deposited unit
A: DNA primase/helicase
B: DNA primase/helicase
C: DNA primase/helicase
D: DNA primase/helicase
E: DNA primase/helicase
F: DNA primase/helicase
H: DNA-directed DNA polymerase
P: Primer
T: Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)474,48323
Polyers471,3559
Non-polymers3,12914
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)39550
ΔGint (kcal/M)-188
Surface area (Å2)118230

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Components

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Protein/peptide , 2 types, 7 molecules ABCDEFH

#1: Protein/peptide
DNA primase/helicase / Gene product 4 / Gp4


Mass: 62734.430 Da / Num. of mol.: 6 / Mutation: E343Q
Source: (gene. exp.) Enterobacteria phage T7 (bacteriophage)
Production host: Escherichia coli (E. coli)
References: UniProt: P03692, Transferases, Transferring phosphorus-containing groups, Nucleotidyltransferases, DNA helicase
#2: Protein/peptide DNA-directed DNA polymerase / DNA polymerase / Gene product 5 / Gp5


Mass: 79703.578 Da / Num. of mol.: 1 / Mutation: D5A, E7A
Source: (gene. exp.) Enterobacteria phage T7 (bacteriophage)
Production host: Escherichia coli (E. coli)
References: UniProt: P00581, DNA-directed DNA polymerase, Hydrolases, Acting on ester bonds, Exodeoxyribonucleases producing 5'-phosphomonoesters

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RNA chain / DNA chain , 2 types, 2 molecules PT

#3: RNA chain Primer


Mass: 1842.206 Da / Num. of mol.: 1 / Source: (synth.) Enterobacteria phage T7 (bacteriophage)
#4: DNA chain Template


Mass: 13402.571 Da / Num. of mol.: 1 / Source: (synth.) Enterobacteria phage T7 (bacteriophage)

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Non-polymers , 3 types, 14 molecules

#5: Chemical
ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 6 / Formula: C10H17N2O14P3 / Thymidine triphosphate
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Formula: Mg / Magnesium
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: gp5 DNA polymerase and two gp4 primase subunits complexed with trx, RNA/DNA hybrid, and incoming dTTP (from gp4-gp5 lagging-strand complex LagS1)
Type: COMPLEX / Entity ID: 1, 2, 3, 4 / Source: RECOMBINANT
Source (natural)Organism: Enterobacteria phage T7 (bacteriophage)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
150 mMTris1
2150 mMpotassium chlorideKCl1
33 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameCategory
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 70745 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 1T8E
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421939
ELECTRON MICROSCOPYf_angle_d0.75129699
ELECTRON MICROSCOPYf_dihedral_angle_d12.41112907
ELECTRON MICROSCOPYf_chiral_restr0.1373291
ELECTRON MICROSCOPYf_plane_restr0.0083661

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