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

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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)Secretion
Map dataSecretion
SampleSubtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).Secretion
SourceLegionella pneumophila (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 40 Å
AuthorsGhosal D / Jeong KC / Chang YW / Vogel JP / Jensen GJ
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.
DateDeposition: Feb 15, 2019 / Header (metadata) release: Feb 15, 2017 / Map release: May 8, 2019 / Update: May 8, 2019

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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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Structure viewerEM map:
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Supplemental images

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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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.8 Å/pix.
x 100 pix.
= 780. Å
7.8 Å/pix.
x 120 pix.
= 936. Å
7.8 Å/pix.
x 100 pix.
= 780. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

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).Secretion
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
Number of components: 1

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Component #1: protein, Subtomogram average of the Legionella pneumophila Dot/Ic...

ProteinName: Subtomogram average of the Legionella pneumophila Dot/Icm type IV secretion system with DotF fused to a superfolder GFP (aligning the OM-complex).Secretion
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
Recombinant expression: No
SourceSpecies: Legionella pneumophila (bacteria) / Strain: Lp02

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionpH: 6.9
Support filmunspecified
VitrificationCryogen name: OTHER

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.2 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C2 (2 fold cyclic) / Number of subtomograms: 254
3D reconstructionSoftware: IMOD / Resolution: 40 Å / Resolution method: FSC 0.143 CUT-OFF

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