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- EMDB-16420: Tomogram of three extended phagophores around SCV -

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Basic information

Entry
Database: EMDB / ID: EMD-16420
TitleTomogram of three extended phagophores around SCV
Map data
Sample
  • Cell: HeLa cells infected with Salmonella
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsMeijing L
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: In situ snapshots along a mammalian selective autophagy pathway.
Authors: Meijing Li / Ishita Tripathi-Giesgen / Brenda A Schulman / Wolfgang Baumeister / Florian Wilfling /
Abstract: Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are ...Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of range from 2 to 5 μm in length and 0.5 to 1.5 μm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged -containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of , or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.
History
DepositionJan 3, 2023-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16420.png

Downloads & links

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Map

FileDownload / File: emd_16420.map.gz / Format: CCP4 / Size: 344.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.56 Å/pix.
x 126 pix.
= 2212.56 Å		17.56 Å/pix.
x 840 pix.
= 14750.399 Å		17.56 Å/pix.
x 854 pix.
= 14996.239 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 17.56 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.5454643 - 0.63378215
Average (Standard dev.)-0.0002587348 (±0.07774087)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin41648
Dimensions840854126
Spacing854840126
CellA: 14996.239 Å / B: 14750.399 Å / C: 2212.5598 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : HeLa cells infected with Salmonella

EntireName: HeLa cells infected with Salmonella
Components
  • Cell: HeLa cells infected with Salmonella

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Supramolecule #1: HeLa cells infected with Salmonella

SupramoleculeName: HeLa cells infected with Salmonella / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE-PROPANE
Cryo protectant10% Glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 15 nA / Focused ion beam - Duration: 20 sec. / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is FEI Aquilos FIB. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 120.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 52

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