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- EMDB-0389: Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: EMDB / ID: EMD-0389
TitleStructure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A)/Trx interacting with primase domains of two gp4 subunits (B and C), with gp4 helicase bound to a DNA fork and dTTP (LagL3)
Map dataT7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
Sample
  • Complex: T7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
    • Protein or peptide: gene product 4 helicase-primase
    • Protein or peptide: gene product 5 DNA polymerase
    • Protein or peptide: Thioredoxin 1
    • DNA: Primer
    • DNA: Template
Biological speciesEnterobacteria phage T7 (virus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
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.
History
DepositionDec 5, 2018-
Header (metadata) releaseFeb 20, 2019-
Map releaseMar 6, 2019-
UpdateMar 6, 2019-
Current statusMar 6, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0389.png

Downloads & links

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Map

FileDownload / File: emd_0389.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationT7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
Voxel sizeX=Y=Z: 1.72 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.056814052 - 0.15194245
Average (Standard dev.)0.00036062105 (±0.0044702804)
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-0.0570.1520.000

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Supplemental data

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Sample components

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Entire : T7 DNA polymerase/Trx interacting with two primase domains (B and...

EntireName: T7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
Components
  • Complex: T7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
    • Protein or peptide: gene product 4 helicase-primase
    • Protein or peptide: gene product 5 DNA polymerase
    • Protein or peptide: Thioredoxin 1
    • DNA: Primer
    • DNA: Template

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Supramolecule #1: T7 DNA polymerase/Trx interacting with two primase domains (B and...

SupramoleculeName: T7 DNA polymerase/Trx interacting with two primase domains (B and C) of T7 helicase (gp4) in complex with a fork DNA substrate and dTTP (from gp4-gp5 lagging-strand complex LagL3)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Enterobacteria phage T7 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: gene product 4 helicase-primase

MacromoleculeName: gene product 4 helicase-primase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterobacteria phage T7 (virus)
SequenceString: MDNSHDSDS VFLYHIPCDN CGSSDGNSLF SDGHTFCYVC EKWTAGNEDT KERASKRKPS G GKPMTYNV WNFGESNGRY SALTARGISK ETCQKAGYWI AKVDGVMYQV ADYRDQNGNI VS QKVRDKD KNFKTTGSHK SDALFGKHLW NGGKKIVVTE GEIDMLTVME ...String:
MDNSHDSDS VFLYHIPCDN CGSSDGNSLF SDGHTFCYVC EKWTAGNEDT KERASKRKPS G GKPMTYNV WNFGESNGRY SALTARGISK ETCQKAGYWI AKVDGVMYQV ADYRDQNGNI VS QKVRDKD KNFKTTGSHK SDALFGKHLW NGGKKIVVTE GEIDMLTVME LQDCKYPVVS LGH GASAAK KTCAANYEYF DQFEQIILMF DMDEAGRKAV EEAAQVLPAG KVRVAVLPCK DANE CHLNG HDREIMEQVW NAGPWIPDGV VSALSLRERI REHLSSEESV GLLFSGCTGI NDKTL GARG GEVIMVTSGS GMGKSTFVRQ QALQWGTAMG KKVGLAMLQE SVEETAEDLI GLHNRV RLR QSDSLKREII ENGKFDQWFD ELFGNDTFHL YDSFAEAETD RLLAKLAYMR SGLGCDV II LDHISIVVSA SGESDERKMI DNLMTKLKGF AKSTGVVLVV ICHLKNPDKG KAHEEGRP V SITDLRGSGA LRQLSDTIIA LERNQQGDMP NLVLVRILKC RFTGDTGIAG YMEYNKETG WLEPSSYSGE EESHSESTDW SNDTDF

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Macromolecule #2: gene product 5 DNA polymerase

MacromoleculeName: gene product 5 DNA polymerase / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Enterobacteria phage T7 (virus)
SequenceString: MIVSAIAANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPAL TKLAKLQLNR EFHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR F GSHALEAW GYRLGEMKGE YKDDFKRMLE EQGEEYVDGM EWWNFNEEMM ...String:
MIVSAIAANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPAL TKLAKLQLNR EFHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR F GSHALEAW GYRLGEMKGE YKDDFKRMLE EQGEEYVDGM EWWNFNEEMM DYNVQDVVVT KA LLEKLLS DKHYFPPEID FTDVGYTTFW SESLEAVDIE HRAAWLLAKQ ERNGFPFDTK AIE ELYVEL AARRSELLRK LTETFGSWYQ PKGGTEMFCH PRTGKPLPKY PRIKTPKVGG IFKK PKNKA QREGREPCEL DTREYVAGAP YTPVEHVVFN PSSRDHIQKK LQEAGWVPTK YTDKG APVV DDEVLEGVRV DDPEKQAAID LIKEYLMIQK RIGQSAEGDK AWLRYVAEDG KIHGSV NPN GAVTGRATHA FPNLAQIPGV RSPYGEQCRA AFGAEHHLDG ITGKPWVQAG IDASGLE LR CLAHFMARFD NGEYAHEILN GDIHTKNQIA AELPTRDNAK TFIYGFLYGA GDEKIGQI V GAGKERGKEL KKKFLENTPA IAALRESIQQ TLVESSQWVA GEQQVKWKRR WIKGLDGRK VHVRSPHAAL NTLLQSAGAL ICKLWIIKTE EMLVEKGLKH GWDGDFAYMA WVHDEIQVGC RTEEIAQVV IETAQEAMRW VGDHWNFRCL LDTEGKMGPN WAICH

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Macromolecule #3: Thioredoxin 1

MacromoleculeName: Thioredoxin 1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
MSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY QGKLTVAKLN IDQNPGTAP KYGIRGIPTL LLFKNGEVAA TKVGALSKGQ LKEFLDANLA

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Macromolecule #4: Primer

MacromoleculeName: Primer / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
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)(DOA)

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Macromolecule #5: Template

MacromoleculeName: Template / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
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)(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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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: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 20298
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1t8e

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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