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- EMDB-11905: In situ subtomogram averaging structure of the type III secretion... -

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Entry
Database: EMDB / ID: EMD-11905
TitleIn situ subtomogram averaging structure of the type III secretion system of yersinia enterocolitica - sorting platformType three secretion system
Map dataSubtomogram average structure of the sorting platform of the type III secretion system of yersinia enterocolitica with C6 symmetry applied, filted to 4 nm resolution.Type three secretion system
Sample
  • Cell: Sorting platform of the Type III secretion system of Yersinia enterocoliticaType three secretion system
Biological speciesYersinia enterocolitica (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsBerger C / Ravelli RBG / Lopez-Iglesias C / Kudryashev M / Diepold A / Peters PJ
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
CitationJournal: J Struct Biol / Year: 2021
Title: Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography.
Authors: Casper Berger / Raimond B G Ravelli / Carmen López-Iglesias / Mikhail Kudryashev / Andreas Diepold / Peter J Peters /
Abstract: Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. ...Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.
History
DepositionOct 28, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateMar 3, 2021-
Current statusMar 3, 2021Processing site: PDBe / Status: Released

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11905.png

Downloads & links

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Map

FileDownload / File: emd_11905.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average structure of the sorting platform of the type III secretion system of yersinia enterocolitica with C6 symmetry applied, filted to 4 nm resolution.
Voxel sizeX=Y=Z: 5.452 Å
Density
Contour LevelBy AUTHOR: 0.32
Minimum - Maximum-5.846176 - 8.15671
Average (Standard dev.)-0.026826719 (±0.44046053)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 1395.712 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.4525.4525.452
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z1395.7121395.7121395.712
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-5.8468.157-0.027

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Supplemental data

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Mask #1

Fileemd_11905_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Subtomogram average structure of the sorting platform of...

Fileemd_11905_additional_1.map
AnnotationSubtomogram average structure of the sorting platform of the type III secretion system of yersinia enterocolitica with C6 symmetry applied.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map used to determine the resolution with C6 symmetry applied

Fileemd_11905_half_map_1.map
Annotationhalf-map used to determine the resolution with C6 symmetry applied
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map used to determine the resolution with C6 symmetry applied

Fileemd_11905_half_map_2.map
Annotationhalf-map used to determine the resolution with C6 symmetry applied
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Sorting platform of the Type III secretion system of Yersinia ent...

EntireName: Sorting platform of the Type III secretion system of Yersinia enterocoliticaType three secretion system
Components
  • Cell: Sorting platform of the Type III secretion system of Yersinia enterocoliticaType three secretion system

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Supramolecule #1: Sorting platform of the Type III secretion system of Yersinia ent...

SupramoleculeName: Sorting platform of the Type III secretion system of Yersinia enterocolitica
type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Yersinia enterocolitica (bacteria) / Strain: E40

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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: 7.4
Details: CElls were grown in RPMI 1640 medium on the grid. 2 ul of RPMI 1640 was applied to the grid just prior to vitrification
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 310 K / Instrument: HOMEMADE PLUNGER
Details: FEI vitrobot modified with a jet-vitrification module and a blotforce feedback mechanism..

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: -0.005 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average exposure time: 24.0 sec. / Average electron dose: 2.86 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 63 / Number images used: 202 / Reference model: averaged from data after manual alignment / Method: manually picked / Software - Name: Dynamo (ver. 1.1.401)
Details: 4x binned tomograms were filtered with EMDeconvolution for manual picking. Coordinates were transformed to extract particles from 2x binned tomograms after CTF correction and without filtering.
CTF correctionSoftware - Name: IMOD (ver. 4) / Details: CTF correction performed with IMOD
Final angle assignmentType: NOT APPLICABLE / Software - Name: Dynamo (ver. 1.1.401)
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo (ver. 1.1.401)
Details: even and odd particles aligned independently after manual alignment
Number subtomograms used: 101
DetailsTilt-series aligned and reconstructed with IMOD with gold fiducials.
FSC plot (resolution estimation)

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