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- EMDB-2717: Electron cryo-microscopy of T7 bacteriophage tail after DNA ejection -

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Basic information

Entry
Database: EMDB / ID: EMD-2717
TitleElectron cryo-microscopy of T7 bacteriophage tail after DNA ejection
Map dataReconstruction of T7 tail after DNA ejection
Sample
  • Sample: T7 tail after DNA ejection
  • Protein or peptide: gp8
  • Protein or peptide: gp11
  • Protein or peptide: gp12
  • Protein or peptide: gp17
  • Protein or peptide: gp10EMD GP10
Keywordsbacteriophage / virus infection / DNA ejection / tail
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsGonzalez-Garcia VA / Pulido-Cid M / Garcia-Doval C / van Raaij MJ / Martin-Benito J / Cuervo A / Carrascosa JL
CitationJournal: J Biol Chem / Year: 2015
Title: Conformational changes leading to T7 DNA delivery upon interaction with the bacterial receptor.
Authors: Verónica A González-García / Mar Pulido-Cid / Carmela Garcia-Doval / Rebeca Bocanegra / Mark J van Raaij / Jaime Martín-Benito / Ana Cuervo / José L Carrascosa /
Abstract: The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly ...The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.
History
DepositionJul 25, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseMar 4, 2015-
UpdateApr 29, 2015-
Current statusApr 29, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image_emd271...

Downloads & links

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Map

FileDownload / File: emd_2717.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of T7 tail after DNA ejection
Voxel sizeX=Y=Z: 5.5 Å
Density
Contour LevelBy AUTHOR: 0.0275 / Movie #1: 0.0275
Minimum - Maximum-0.10356901 - 0.1457787
Average (Standard dev.)0.00132477 (±0.0233259)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.55.55.5
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.1040.1460.001

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Supplemental data

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Sample components

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Entire : T7 tail after DNA ejection

EntireName: T7 tail after DNA ejection
Components
  • Sample: T7 tail after DNA ejection
  • Protein or peptide: gp8
  • Protein or peptide: gp11
  • Protein or peptide: gp12
  • Protein or peptide: gp17
  • Protein or peptide: gp10EMD GP10

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Supramolecule #1000: T7 tail after DNA ejection

SupramoleculeName: T7 tail after DNA ejection / type: sample / ID: 1000
Oligomeric state: Oligomer build of 5 components: gp8 (12mer), gp11 (12mer), gp12 (6mer), gp17 (3mer), gp10 (n/a)
Number unique components: 5

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Macromolecule #1: gp8

MacromoleculeName: gp8 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #2: gp11

MacromoleculeName: gp11 / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #3: gp12

MacromoleculeName: gp12 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #4: gp17

MacromoleculeName: gp17 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #5: gp10

MacromoleculeName: gp10 / type: protein_or_peptide / ID: 5 / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus)

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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

Concentration0.1 mg/mL
BufferpH: 7.8
Details: 50 mM TrisHCl, 10 mM MgCl2, 100 mM NaCl, 0.25mg/ml LPS from E. coli EH 100 Ra mutant
GridDetails: R2/2 Quantifoil coated with a thin carbon layer
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC
Method: Samples were applied to grids for 1 minute, blotted, and plunged into liquid ethane.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 108696
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
DateOct 11, 2012
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 2 µm / Number real images: 463 / Average electron dose: 10 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 4
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: XMIPP, EMAN / Details: Final map was calculated from 3 averaged data sets / Number images used: 1781
DetailsThe particles were manually selected

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