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- EMDB-0382: Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: EMDB / ID: EMD-0382
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 (LagS3)
Map dataStructure of gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
Sample
  • Complex: gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
    • Protein or peptide: DNA primase/helicase
    • Protein or peptide: DNA-directed DNA polymeraseDNA polymerase
    • RNA: Primer
    • DNA: Template
  • Ligand: ZINC ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / DNA primase activity / 5'-3' DNA helicase activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / 3'-5' exonuclease activity / DNA helicase activity / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...DNA synthesis involved in DNA replication / DNA exonuclease activity / DNA primase activity / 5'-3' DNA helicase activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / 3'-5' exonuclease activity / DNA helicase activity / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding / DNA helicase / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / identical protein binding / metal ion binding
Similarity search - Function
Bacteriophage T7 DNA helicase/primase / : / Bacteriophage T7 DNA helicase/primase, N-terminal a+b fold / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Zinc-binding domain of primase-helicase / Zinc-binding domain of primase-helicase / DNA-directed DNA polymerase T7 / Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / Toprim-like ...Bacteriophage T7 DNA helicase/primase / : / Bacteriophage T7 DNA helicase/primase, N-terminal a+b fold / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Zinc-binding domain of primase-helicase / Zinc-binding domain of primase-helicase / DNA-directed DNA polymerase T7 / Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / Toprim-like / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / TOPRIM / 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 / Toprim domain profile. / TOPRIM domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA-directed DNA polymerase / DNA helicase/primase
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsGao Y / Fox T / Val N / Yang W
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
DepositionDec 4, 2018-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6n9x
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0382.png

Downloads & links

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Map

FileDownload / File: emd_0382.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.082968704 - 0.15626752
Average (Standard dev.)0.0002027223 (±0.0031144926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0830.1560.000

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Supplemental data

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Sample components

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

EntireName: gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
Components
  • Complex: gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
    • Protein or peptide: DNA primase/helicase
    • Protein or peptide: DNA-directed DNA polymeraseDNA polymerase
    • RNA: Primer
    • DNA: Template
  • Ligand: ZINC ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: gp5 DNA polymerase binding to B and C subunits of gp4 primase-hel...

SupramoleculeName: gp5 DNA polymerase binding to B and C subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS3)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Enterobacteria phage T7 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: DNA primase/helicase

MacromoleculeName: DNA primase/helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 62.73443 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDNSHDSDSV FLYHIPCDNC GSSDGNSLFS DGHTFCYVCE KWTAGNEDTK ERASKRKPSG GKPMTYNVWN FGESNGRYSA LTARGISKE TCQKAGYWIA KVDGVMYQVA DYRDQNGNIV SQKVRDKDKN FKTTGSHKSD ALFGKHLWNG GKKIVVTEGE I DMLTVMEL ...String:
MDNSHDSDSV FLYHIPCDNC GSSDGNSLFS DGHTFCYVCE KWTAGNEDTK ERASKRKPSG GKPMTYNVWN FGESNGRYSA LTARGISKE TCQKAGYWIA KVDGVMYQVA DYRDQNGNIV SQKVRDKDKN FKTTGSHKSD ALFGKHLWNG GKKIVVTEGE I DMLTVMEL QDCKYPVVSL GHGASAAKKT CAANYEYFDQ FEQIILMFDM DEAGRKAVEE AAQVLPAGKV RVAVLPCKDA NE CHLNGHD REIMEQVWNA GPWIPDGVVS ALSLRERIRE HLSSEESVGL LFSGCTGIND KTLGARGGEV IMVTSGSGMG KST FVRQQA LQWGTAMGKK VGLAMLQESV EETAEDLIGL HNRVRLRQSD SLKREIIENG KFDQWFDELF GNDTFHLYDS FAEA ETDRL LAKLAYMRSG LGCDVIILDH ISIVVSASGE SDERKMIDNL MTKLKGFAKS TGVVLVVICH LKNPDKGKAH EEGRP VSIT DLRGSGALRQ LSDTIIALER NQQGDMPNLV LVRILKCRFT GDTGIAGYME YNKETGWLEP SSYSGEEESH SESTDW SND TDF

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Macromolecule #2: DNA-directed DNA polymerase

MacromoleculeName: DNA-directed DNA polymerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 79.703578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIVSAIAANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPALT KLAKLQLNRE FHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR FGSHALEAWG YRLGEMKGEY KDDFKRMLEE QGEEYVDGME W WNFNEEMM ...String:
MIVSAIAANA 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

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Macromolecule #3: Primer

MacromoleculeName: Primer / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 1.842206 KDa
SequenceString:
ACCAG(DOC)

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Macromolecule #4: Template

MacromoleculeName: Template / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 13.402571 KDa
SequenceString:
(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)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: THYMIDINE-5'-TRIPHOSPHATE

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

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
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


Details: 1T8E, 1NUI
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 38544
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1t8e

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6n9x:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS3)

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