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- EMDB-15549: In situ cryo-electron tomogram of a bulk autophagy phagophore in ... -
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Open data
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Basic information
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Title | In situ cryo-electron tomogram of a bulk autophagy phagophore in S. cerevisiae #4 | |||||||||
![]() | In situ yeast phagophore #4 | |||||||||
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![]() | Autophagy / phagophore / fusion / yeast / bulk / degradation / RIBOSOME | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | electron tomography / cryo EM | |||||||||
![]() | Capitanio C / Bieber A / Erdmann PS / Schulman BA / Baumeister W / Wilfling F | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1 GB | ![]() | |
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Header (meta data) | ![]() ![]() | 12 KB 12 KB | Display Display | ![]() |
Images | ![]() | 254.9 KB | ||
Filedesc metadata | ![]() | 4.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | In situ yeast phagophore #4 | ||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 14.08 Å | ||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : In situ cryo-electron tomogram of a bulk autophagy phagophore in ...
Entire | Name: In situ cryo-electron tomogram of a bulk autophagy phagophore in S. cerevisiae #4 |
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Components |
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-Supramolecule #1: In situ cryo-electron tomogram of a bulk autophagy phagophore in ...
Supramolecule | Name: In situ cryo-electron tomogram of a bulk autophagy phagophore in S. cerevisiae #4 type: cell / ID: 1 / Parent: 0 Details: Phagophore in the cytoplasm next to the vacuole. The position of the phagophore corresponds to eGFP-Atg8 signal detected by cryo-fluorescence microscopy. |
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Source (natural) | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | electron tomography |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 7 |
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Grid | Model: Quantifoil / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV |
Details | S. cerevisiae. 2 hours starvation in SD-N. |
Sectioning | Focused 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
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.75 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Software - Name: ![]() |
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