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- EMDB-0365: Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: EMDB / ID: 0365
TitleStructure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes)
Map dataD5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
SampleD5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
  • DNA-directed DNA polymeraseDNA polymerase
  • TrxA
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 2
Function / homologyThioredoxin / DNA polymerase family A / Thioredoxin-like superfamily / DNA-directed DNA polymerase T7 / DNA-directed DNA polymerase, family A, conserved site / Thioredoxin, conserved site / Thioredoxin domain / Ribonuclease H-like superfamily / DNA polymerase A / DNA-directed DNA polymerase, family A, palm domain ...Thioredoxin / DNA polymerase family A / Thioredoxin-like superfamily / DNA-directed DNA polymerase T7 / DNA-directed DNA polymerase, family A, conserved site / Thioredoxin, conserved site / Thioredoxin domain / Ribonuclease H-like superfamily / DNA polymerase A / DNA-directed DNA polymerase, family A, palm domain / Ribonuclease H superfamily / Thioredoxin family active site. / Thioredoxin / DNA polymerase family A signature. / Thioredoxin domain profile. / viral DNA genome replication / Hydrolases, Acting on ester bonds, Exodeoxyribonucleases producing 5'-phosphomonoesters / glycerol ether metabolic process / protein disulfide oxidoreductase activity / 3'-5' exonuclease activity / DNA-dependent DNA replication / cell redox homeostasis / cell / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / DNA-directed DNA polymerase / TrxA
Function and homology information
SourceEnterobacteria phage T7 (bacteriophage) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 4.5 Å resolution
AuthorsGao Y / Fox T / Val N / 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: 6n7w

SummaryFull reportAbout validation report
DateDeposition: Nov 28, 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: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6n7w
  • Surface level: 0.07
  • 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_0365.map.gz (map file in CCP4 format, 55297 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
240 pix
1.72 Å/pix.
= 412.8 Å
240 pix
1.72 Å/pix.
= 412.8 Å
240 pix
1.72 Å/pix.
= 412.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.72 Å
Density
Contour Level:0.07 (by author), 0.07 (movie #1):
Minimum - Maximum-0.10222357 - 0.29595906
Average (Standard dev.)0.00012666905 (0.0041107354)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions240240240
Origin0.00.00.0
Limit239.0239.0239.0
Spacing240240240
CellA=B=C: 412.80002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.721.721.72
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z412.800412.800412.800
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1020.2960.000

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Supplemental data

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Sample components

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Entire D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, an...

EntireName: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
Number of components: 7

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Component #1: protein, D5AE7A mutant gp5 DNA polymerase complexed with trx, for...

ProteinName: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
Recombinant expression: No
SourceSpecies: Enterobacteria phage T7 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: 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 #3: protein, TrxA

ProteinName: TrxA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.818582 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: nucleic-acid, DNA (25-MER)

Nucleic-acidName: DNA (25-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DT)(DA)(DC)(DA)(DA)(DC)(DT)(DT) (DG)(DA)(DC)(DG)(DA)(DC)(DA)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(2DA)
MassTheoretical: 7.716011 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)

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Component #5: nucleic-acid, DNA (77-MER)

Nucleic-acidName: DNA (77-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(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)(DA)(DC) (DG)(DG)(DA)(DG)(DT)(DC)(DG)(DT)(DT)(DT) (DC)(DG)(DA)(DC)(DT)(DC)(DC)(DG)(DT)(DT) (DA)(DT)(DC)(DA)(DC)(DG)(DC)(DT)(DA)(DT) (DG)(DT)(DC)(DG)(DT)(DC)(DA)(DA)(DG)(DT) (DT)(DG)(DT)(DA)(DC)(DC)
MassTheoretical: 23.278844 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)

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Component #6: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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

LigandName: THYMIDINE-5'-TRIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.482168 kDa

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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 / Number of projections: 129003
3D reconstructionSoftware: RELION / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 1T8E
Output model

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