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- EMDB-0381: Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/... -

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Entry
Database: EMDB / ID: 0381
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 (LagS2)
Map dataStructure of gp5 DNA polymerase binding to A and B subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS2)
Samplegp5 DNA polymerase binding to A and B subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS2)
  • DNA primase/helicase
  • DNA-directed DNA polymeraseDNA polymerase
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 3
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
SourceEnterobacteria phage T7 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / 4 Å resolution
AuthorsGao Y / Cui Y / Zhou Z / Yang W
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 ReportPDB-ID: 6n9w

SummaryFull reportAbout validation report
DateDeposition: Dec 4, 2018 / Header (metadata) release: Feb 20, 2019 / Map release: Mar 6, 2019 / Last update: Mar 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6n9w
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0381.map.gz (map file in CCP4 format, 256001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
400 pix
0.86 Å/pix.
= 344. Å
400 pix
0.86 Å/pix.
= 344. Å
400 pix
0.86 Å/pix.
= 344. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density
Contour Level:0.4 (by author), 0.4 (movie #1):
Minimum - Maximum-0.5765288 - 1.3659258
Average (Standard dev.)-0.0020261337 (0.044603284)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions400400400
Origin0.00.00.0
Limit399.0399.0399.0
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z344.000344.000344.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.5771.366-0.002

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Supplemental data

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Sample components

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Entire gp5 DNA polymerase binding to A and B subunits of gp4 primase-hel...

EntireName: gp5 DNA polymerase binding to A and B subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS2)
Number of components: 8

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Component #1: protein, gp5 DNA polymerase binding to A and B subunits of gp4 pr...

ProteinName: gp5 DNA polymerase binding to A and B subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS2)
Recombinant expression: No
SourceSpecies: Enterobacteria phage T7 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, DNA primase/helicase

ProteinName: DNA primase/helicase / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 62.73443 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, DNA-directed DNA polymerase

ProteinName: DNA-directed DNA polymeraseDNA polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 79.805625 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: nucleic-acid, Primer

Nucleic-acidName: Primer / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
ACCAG(DOC)
MassTheoretical: 1.842206 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)

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Component #5: nucleic-acid, Template

Nucleic-acidName: Template / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DT)(DA)(DG)(DC)(DT)(DG) (DG)(DT)(DC)(DA)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)
MassTheoretical: 13.402571 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)

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Component #6: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #7: ligand, THYMIDINE-5'-TRIPHOSPHATE

LigandName: THYMIDINE-5'-TRIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.482168 kDa

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Component #8: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 295 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 4 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 38760
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 1E0J
Output model

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