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- EMDB-2419: 3-dimensional structure of the toxin-delivery particle antifeedin... -

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Basic information

Entry
Database: EMDB / ID: EMD-2419
Title3-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila
Map dataReconstruction of the full Afp particle
Sample
  • Sample: Antifeeding prophage of Serratia entomophila
  • Protein or peptide: Antifeeding prophage
Keywordstube-baseplate complex / tailocin / type VI secretion system / helical sheath / phage tail-like
Biological speciesSerratia entomophila (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsHeymann JB / Bartho JD / Rybakova D / Venugopal HP / Winkler DC / Sen A / Hurst MRH / Mitra AK
CitationJournal: J Biol Chem / Year: 2013
Title: Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila.
Authors: J Bernard Heymann / Joseph D Bartho / Daria Rybakova / Hari P Venugopal / Dennis C Winkler / Anindito Sen / Mark R H Hurst / Alok K Mitra /
Abstract: The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of ...The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems. Cryo-electron micrographs of tilted Afp specimens (up to 60 degrees) were analyzed to determine the correct cyclic symmetry to overcome the limitation imposed by exclusively side views in nominally untilted specimens. An asymmetric reconstruction shows clear 6-fold cyclic symmetry contrary to a previous conclusion of 4-fold symmetry based on analysis of only the preferred side views (Sen, A., Rybakova, D., Hurst, M. R., and Mitra, A. K. (2010) J. Bacteriol. 192, 4522-4525). Electron tomography of negatively stained Afp revealed right-handed helical striations in many of the particles, establishing the correct hand. Higher quality micrographs of untilted specimens were processed to produce a reconstruction at 2.0-nm resolution with imposed 6-fold symmetry. The helical parameters of the sheath were determined to be 8.14 nm for the subunit rise along and 40.5° for the rotation angle around the helix. The sheath is similar to that of the T4 phage tail but with a different arrangement of the subdomain of the polymerizing sheath protein(s). The central tube is similar to the diameter and axial width of the Hcp1 hexamer of P. aeruginosa type VI secretion system. The tube extends through the baseplate into a needle resembling the "puncture device" of the T4 tail. The tube contains density that may be the toxin and/or a length-determining protein.
History
DepositionJul 18, 2013-
Header (metadata) releaseJul 31, 2013-
Map releaseAug 7, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2419.png

Downloads & links

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Map

FileDownload / File: emd_2419.map.gz / Format: CCP4 / Size: 21.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the full Afp particle
Voxel sizeX=Y=Z: 3.07 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-6.86292648 - 9.212231640000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-200
Dimensions120120400
Spacing120120400
CellA: 368.4 Å / B: 368.4 Å / C: 1228.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.073.073.07
M x/y/z120120400
origin x/y/z0.0000.0000.000
length x/y/z368.400368.4001228.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-60-60-200
NC/NR/NS120120400
D min/max/mean-6.8639.212-0.000

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Supplemental data

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Sample components

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Entire : Antifeeding prophage of Serratia entomophila

EntireName: Antifeeding prophage of Serratia entomophila
Components
  • Sample: Antifeeding prophage of Serratia entomophila
  • Protein or peptide: Antifeeding prophage

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Supramolecule #1000: Antifeeding prophage of Serratia entomophila

SupramoleculeName: Antifeeding prophage of Serratia entomophila / type: sample / ID: 1000 / Details: Afp particle produced from 18 ORF products / Number unique components: 1

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Macromolecule #1: Antifeeding prophage

MacromoleculeName: Antifeeding prophage / type: protein_or_peptide / ID: 1 / Name.synonym: Afp
Details: Product of the expression of 18 ORF's of the pADAP plasmid of Serratia entomophila
Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Serratia entomophila (bacteria)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
Recombinant plasmid: pAF6-21, pASANFA1

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 25 mM TBS, 130 mM NaCl
StainingType: NEGATIVE / Details: None (cryo)
GridDetails: Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: HOMEMADE PLUNGER
Method: Glow-discharge grid in amyl amine atmosphere Apply 5 ul sample, 4C, humidity 90% Blot Plunge

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 41423 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
DateFeb 11, 2013
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 113 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each particle, phase flipping, baseline correction
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: Bsoft / Details: Final map filtered by FSC curve / Number images used: 1848
FSC plot (resolution estimation)

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