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- EMDB-0566: Subtomogram average of the Legionella pneumophila Dot/Icm type IV... -

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Basic information

Entry
Database: EMDB / ID: EMD-0566
TitleSubtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex)
Map dataSubtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).
Sample
  • Complex: Subtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).
Biological speciesLegionella pneumophila (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsGhosal D / Jeong KC / Chang YW / Vogel JP / Jensen GJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01AI127401 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01AI48052 United States
CitationJournal: Nat Microbiol / Year: 2019
Title: Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS.
Authors: Debnath Ghosal / Kwangcheol C Jeong / Yi-Wei Chang / Jacob Gyore / Lin Teng / Adam Gardner / Joseph P Vogel / Grant J Jensen /
Abstract: Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion ...Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.
History
Header (metadata) releaseFeb 15, 2017-
DepositionFeb 15, 2019-
Map releaseMay 8, 2019-
UpdateJul 3, 2019-
Current statusJul 3, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.51
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.51
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0566.png

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Map

FileDownload / File: emd_0566.map.gz / Format: CCP4 / Size: 4.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).
Voxel sizeX=Y=Z: 7.8 Å
Density
Contour LevelBy AUTHOR: 0.51 / Movie #1: 0.51
Minimum - Maximum-0.00000011920929 - 1.0
Average (Standard dev.)0.38468668 (±0.18937564)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120100100
Spacing100120100
CellA: 780.0 Å / B: 936.0 Å / C: 780.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.87.87.8
M x/y/z100120100
origin x/y/z0.0000.0000.000
length x/y/z780.000936.000780.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100120100
D min/max/mean-0.0001.0000.385

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Supplemental data

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Sample components

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Entire : Subtomogram average of the Legionella pneumophila Dot/Icm type IV...

EntireName: Subtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).
Components
  • Complex: Subtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).

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Supramolecule #1: Subtomogram average of the Legionella pneumophila Dot/Icm type IV...

SupramoleculeName: Subtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).
type: complex / ID: 1 / Parent: 0
Details: We used electron cryotomography and subtomogram averaging to visualize L. pneumophila Dot/Icm (DotF-GFP fusion) complex directly in intact, frozen-hydrated bacterial cells
Source (natural)Organism: Legionella pneumophila (bacteria) / Strain: Lp02

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 6.9
GridDetails: unspecified
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMOD / Number subtomograms used: 254
ExtractionNumber tomograms: 75 / Number images used: 254 / Software - Name: PEET
Final angle assignmentType: OTHER

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