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- EMDB-8565: Cryo-EM structure of bacteriphage T7 replisome -

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Basic information

Entry
Database: EMDB / ID: EMD-8565
TitleCryo-EM structure of bacteriphage T7 replisome
Map dataCryo-EM structure of bacteriophage T7 replisome
Sample
  • Complex: Bacteriophage T7 replisome. A complex of DNA primase-helicase with two molecules of DNA polymerase
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / DNA primase activity / primosome complex / 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 / primosome complex / 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 / double-strand break repair / single-stranded DNA binding / 5'-3' DNA helicase activity / 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: 13.8 Å
AuthorsKulczyk AW
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis.
Authors: Arkadiusz W Kulczyk / Arne Moeller / Peter Meyer / Piotr Sliz / Charles C Richardson /
Abstract: We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, ...We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.
History
DepositionJan 20, 2017-
Header (metadata) releaseFeb 15, 2017-
Map releaseFeb 22, 2017-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8565.png

Downloads & links

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Map

FileDownload / File: emd_8565.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of bacteriophage T7 replisome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 300 pix.
= 300. Å		1 Å/pix.
x 300 pix.
= 300. Å		1 Å/pix.
x 300 pix.
= 300. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0015 / Movie #1: 0.0015
Minimum - Maximum-0.001991717 - 0.017968316
Average (Standard dev.)0.000089346104 (±0.0006598306)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 300.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z300.000300.000300.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-0.0020.0180.000

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Supplemental data

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Sample components

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Entire : Bacteriophage T7 replisome. A complex of DNA primase-helicase wit...

EntireName: Bacteriophage T7 replisome. A complex of DNA primase-helicase with two molecules of DNA polymerase
Components
  • Complex: Bacteriophage T7 replisome. A complex of DNA primase-helicase with two molecules of DNA polymerase

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Supramolecule #1: Bacteriophage T7 replisome. A complex of DNA primase-helicase wit...

SupramoleculeName: Bacteriophage T7 replisome. A complex of DNA primase-helicase with two molecules of DNA polymerase
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Enterobacteria phage T7 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 650 kDa/nm

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 5.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 2.0 µm / Illumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4) / Details: CTFFIND4
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 13.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 79519
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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