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- EMDB-5713: Electron microscopy of negatively-stained gp12 tubular protein of... -

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Basic information

Entry
Database: EMDB / ID: EMD-5713
TitleElectron microscopy of negatively-stained gp12 tubular protein of T7 bacteriophage
Map dataReconstruction of gp12 tubular protein of T7 bacteriophage
Sample
  • Sample: T7 tail tubular protein gp12
  • Protein or peptide: gp12
KeywordsBacteriophage / DNA ejection / tail structure / tail tubular protein
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsCuervo A / Pulido-Cid M / Chagoyen M / Arranz R / Gonzalez-Garcia VA / Garcia-Doval C / Caston JR / Valpuesta JM / van Raaij MJ / Martin-Benito J / Carrascosa JL
CitationJournal: J Biol Chem / Year: 2013
Title: Structural characterization of the bacteriophage T7 tail machinery.
Authors: Ana Cuervo / Mar Pulido-Cid / Mónica Chagoyen / Rocío Arranz / Verónica A González-García / Carmela Garcia-Doval / José R Castón / José M Valpuesta / Mark J van Raaij / Jaime Martín- ...Authors: Ana Cuervo / Mar Pulido-Cid / Mónica Chagoyen / Rocío Arranz / Verónica A González-García / Carmela Garcia-Doval / José R Castón / José M Valpuesta / Mark J van Raaij / Jaime Martín-Benito / José L Carrascosa /
Abstract: Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well ...Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.
History
DepositionJul 2, 2013-
Header (metadata) releaseSep 11, 2013-
Map releaseSep 11, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.109022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.109022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_5713.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of gp12 tubular protein of T7 bacteriophage
Voxel sizeX=Y=Z: 4.14 Å
Density
Contour LevelBy AUTHOR: 0.109022 / Movie #1: 0.109022
Minimum - Maximum-0.12215641 - 0.27921242
Average (Standard dev.)0.00071014 (±0.01027719)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.144.144.14
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z331.200331.200331.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.1220.2790.001

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Supplemental data

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Sample components

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Entire : T7 tail tubular protein gp12

EntireName: T7 tail tubular protein gp12
Components
  • Sample: T7 tail tubular protein gp12
  • Protein or peptide: gp12

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Supramolecule #1000: T7 tail tubular protein gp12

SupramoleculeName: T7 tail tubular protein gp12 / type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 1
Molecular weightTheoretical: 90 KDa

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Macromolecule #1: gp12

MacromoleculeName: gp12 / type: protein_or_peptide / ID: 1 / Name.synonym: Tail tubular protein / Details: The protein was cloned with a His-tag. / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage T7 (virus) / synonym: T7 Bacteriophage
Molecular weightTheoretical: 90 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C41 / Recombinant plasmid: pRSET-B

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.8 / Details: 50mM Tris-HCl, 10mM MgCl2, 100 mM NaCl
StainingType: NEGATIVE
Details: Samples were applied to grids for 1 min, washed with water, blotted, and stained for 1 min in uranyl acetate.
GridDetails: 400 mesh cupper grid coated with a carbon layer, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 108696 / Illumination 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: PHILIPS ROTATION HOLDER
DateNov 8, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 181 / 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 reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: XMIPP, SPIDER, EMAN / Number images used: 640

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