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- EMDB-15547: In situ cryo-electron tomogram of a bulk autophagy autophagosome ... -

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Basic information

Entry
Database: EMDB / ID: EMD-15547
TitleIn situ cryo-electron tomogram of a bulk autophagy autophagosome fusing with the vacuole in S. cerevisiae #1
Map dataFusion of autophagosome with the vacuole, in situ tomogram in S. cerevisiae
Sample
  • Cell: In situ cryo-electron tomogram of a bulk autophagy autophagosome fusing with the vacuole in S. cerevisiae #1
KeywordsAutophagy / phagophore / fusion / yeast / bulk / degradation / RIBOSOME
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsBieber A / Capitanio C / Erdmann PS / Schulman BA / Baumeister W / Wilfling F
Funding support United States, 1 items
OrganizationGrant numberCountry
Other privateASAP-000282 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: In situ structural analysis reveals membrane shape transitions during autophagosome formation.
Authors: Anna Bieber / Cristina Capitanio / Philipp S Erdmann / Fabian Fiedler / Florian Beck / Chia-Wei Lee / Delong Li / Gerhard Hummer / Brenda A Schulman / Wolfgang Baumeister / Florian Wilfling /
Abstract: Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly ...Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
History
DepositionAug 8, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15547.png

Downloads & links

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Map

FileDownload / File: emd_15547.map.gz / Format: CCP4 / Size: 821.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFusion of autophagosome with the vacuole, in situ tomogram in S. cerevisiae
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
14.08 Å/pix.
x 250 pix.
= 3520. Å		14.08 Å/pix.
x 928 pix.
= 13066.24 Å		14.08 Å/pix.
x 928 pix.
= 13066.24 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: 14.08 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-70.390209999999996 - 42.146909999999998
Average (Standard dev.)0.6576484 (±6.128873)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-125
Dimensions928928250
Spacing928928250
CellA: 13066.24 Å / B: 13066.24 Å / C: 3520.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : In situ cryo-electron tomogram of a bulk autophagy autophagosome ...

EntireName: In situ cryo-electron tomogram of a bulk autophagy autophagosome fusing with the vacuole in S. cerevisiae #1
Components
  • Cell: In situ cryo-electron tomogram of a bulk autophagy autophagosome fusing with the vacuole in S. cerevisiae #1

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Supramolecule #1: In situ cryo-electron tomogram of a bulk autophagy autophagosome ...

SupramoleculeName: In situ cryo-electron tomogram of a bulk autophagy autophagosome fusing with the vacuole in S. cerevisiae #1
type: cell / ID: 1 / Parent: 0
Details: Autophagosome fusing with the vacuole. The position of the autophagosome corresponds to mCherry-Atg8 signal detected by cryo-fluorescence microscopy.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: DF5

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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
GridModel: Quantifoil / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsS. cerevisiae. 2 hours starvation in SD-N.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.3 / Focused ion beam - Duration: 2400 / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 7000 / Focused ion beam - Final thickness: 150
Focused ion beam - Details: Correlative FIB-milling. The lamella was prepared in the location of fluorescence signal.. The value given for _em_focused_ion_beam.instrument is Other. This is not in a ...Focused ion beam - Details: Correlative FIB-milling. The lamella was prepared in the location of fluorescence signal.. The value given for _em_focused_ion_beam.instrument is Other. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3712 pixel / Digitization - Dimensions - Height: 3712 pixel / Number real images: 60 / Average exposure time: 2.0 sec. / Average electron dose: 120.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 55

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