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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: EMD-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)
Sample
  • Complex: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
    • Protein or peptide: DNA-directed DNA polymerase
    • Protein or peptide: TrxA
    • DNA: DNA (25-MER)
    • DNA: DNA (77-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
KeywordsDNA polymerase / helicase / DNA replication / replisome / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / double-strand break repair ...DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / double-strand break repair / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / cytoplasm / cytosol
Similarity search - Function
DNA-directed DNA polymerase T7 / Thioredoxin / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Thioredoxin / Thioredoxin, conserved site ...DNA-directed DNA polymerase T7 / Thioredoxin / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA-directed DNA polymerase / Thioredoxin 1 / Thioredoxin
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsGao Y / Fox T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)1S10RR23057 United 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.
History
DepositionNov 28, 2018-
Header (metadata) releaseFeb 20, 2019-
Map releaseMar 6, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • 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
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0365.png

Downloads & links

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Map

FileDownload / File: emd_0365.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationD5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.10222357 - 0.29595906
Average (Standard dev.)0.00012666905 (±0.0041107354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 412.80002 Å
α=β=γ: 90.0 °

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
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)
Components
  • Complex: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
    • Protein or peptide: DNA-directed DNA polymerase
    • Protein or peptide: TrxA
    • DNA: DNA (25-MER)
    • DNA: DNA (77-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, an...

SupramoleculeName: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #1: DNA-directed DNA polymerase

MacromoleculeName: DNA-directed DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 79.805625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIVSDIEANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPALT KLAKLQLNRE FHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR FGSHALEAWG YRLGEMKGEY KDDFKRMLEE QGEEYVDGME W WNFNEEMM ...String:
MIVSDIEANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPALT KLAKLQLNRE FHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR FGSHALEAWG YRLGEMKGEY KDDFKRMLEE QGEEYVDGME W WNFNEEMM DYNVQDVVVT KALLEKLLSD KHYFPPEIDF TDVGYTTFWS ESLEAVDIEH RAAWLLAKQE RNGFPFDTKA IE ELYVELA ARRSELLRKL TETFGSWYQP KGGTEMFCHP RTGKPLPKYP RIKTPKVGGI FKKPKNKAQR EGREPCELDT REY VAGAPY TPVEHVVFNP SSRDHIQKKL QEAGWVPTKY TDKGAPVVDD EVLEGVRVDD PEKQAAIDLI KEYLMIQKRI GQSA EGDKA WLRYVAEDGK IHGSVNPNGA VTGRATHAFP NLAQIPGVRS PYGEQCRAAF GAEHHLDGIT GKPWVQAGID ASGLE LRCL AHFMARFDNG EYAHEILNGD IHTKNQIAAE LPTRDNAKTF IYGFLYGAGD EKIGQIVGAG KERGKELKKK FLENTP AIA ALRESIQQTL VESSQWVAGE QQVKWKRRWI KGLDGRKVHV RSPHAALNTL LQSAGALICK LWIIKTEEML VEKGLKH GW DGDFAYMAWV HDEIQVGCRT EEIAQVVIET AQEAMRWVGD HWNFRCLLDT EGKMGPNWAI CH

UniProtKB: DNA-directed DNA polymerase

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Macromolecule #2: TrxA

MacromoleculeName: TrxA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.818582 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAA TKVGALSKGQ LKEFLDANLA

UniProtKB: Thioredoxin

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Macromolecule #3: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 7.716011 KDa
SequenceString:
(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)

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Macromolecule #4: DNA (77-MER)

MacromoleculeName: DNA (77-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 23.278844 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMpotassium chlorideKCl
3.0 mMDTT
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1t8e

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 129003
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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Atomic model buiding 1

Initial modelPDB ID:

1t8e


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6n7w:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes)

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