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- EMDB-13682: In situ subtomogram average of autophagosome-associated protein f... -

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Basic information

Entry
Database: EMDB / ID: EMD-13682
TitleIn situ subtomogram average of autophagosome-associated protein filament
Map dataAutophagosome-associated protein filament
Sample
  • Cell: Autophagosome-associated protein filament inside poliovirus-infected human cells.
Keywordsvirus / autophagy / membrane / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 18.5 Å
AuthorsDahmane S / Morado DR / Carlson L-A
Funding support Sweden, 3 items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission795892 Sweden
Human Frontier Science Program (HFSP)CDA00047/2017-C Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nat Commun / Year: 2022
Title: Membrane-assisted assembly and selective secretory autophagy of enteroviruses.
Authors: Selma Dahmane / Adeline Kerviel / Dustin R Morado / Kasturika Shankar / Björn Ahlman / Michael Lazarou / Nihal Altan-Bonnet / Lars-Anders Carlson /
Abstract: Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome ...Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.
History
DepositionOct 6, 2021-
Header (metadata) releaseOct 19, 2022-
Map releaseOct 19, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13682.png

Downloads & links

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Map

FileDownload / File: emd_13682.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAutophagosome-associated protein filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.29 Å/pix.
x 100 pix.
= 429. Å		4.29 Å/pix.
x 100 pix.
= 429. Å		4.29 Å/pix.
x 100 pix.
= 429. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.29 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.06239607 - 0.10313908
Average (Standard dev.)-0.00005403114 (±0.0073031676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 429.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map

Fileemd_13682_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unmasked half map

Fileemd_13682_half_map_1.map
Annotationunmasked half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unmasked half map

Fileemd_13682_half_map_2.map
Annotationunmasked half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Autophagosome-associated protein filament inside poliovirus-infec...

EntireName: Autophagosome-associated protein filament inside poliovirus-infected human cells.
Components
  • Cell: Autophagosome-associated protein filament inside poliovirus-infected human cells.

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Supramolecule #1: Autophagosome-associated protein filament inside poliovirus-infec...

SupramoleculeName: Autophagosome-associated protein filament inside poliovirus-infected human cells.
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
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE
DetailsCryo-FIB lamellas of HeLa cells grown on EM-grids and infected with poliovirus type 1.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 33000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 2.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 16 / Number images used: 55376
Final angle assignmentType: ANGULAR RECONSTITUTION / Software: (Name: subTOM, Dynamo)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: subTOM / Number subtomograms used: 141855

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