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- EMDB-57826: In situ cryo-electron tomogram of phagophore expansion in Atg2-PM... -

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Basic information

Entry
Database: EMDB / ID: EMD-57826
TitleIn situ cryo-electron tomogram of phagophore expansion in Atg2-PM4 mutant S. cerevisiae #3
Map dataphagophore in direct contact with the phospholipid monolayer at the surface of a lipid droplet
Sample
  • Cell: Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed under native promoters from plasmids while the native gene copies were knocked out
KeywordsAtg2 / phagophore expansion / autophagosome biogenesis / LIPID TRANSPORT
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
Methodelectron tomography / cryo EM
AuthorsOrtmann de Percin Northumberland C / Licheva M / Dabrowski R / Gomez-Sanchez R / Berkamp S / Schonnenbeck P / Graef M / Kraft C / Sachse C
Funding support Germany, European Union, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)450216812 Germany
German Research Foundation (DFG)409673687 Germany
German Research Foundation (DFG)403222702 Germany
European Research Council (ERC)769065European Union
CitationJournal: J Cell Sci / Year: 2026
Title: In situ visualization of autophagy reveals the presence of vesicles at the expanding phagophore rim.
Authors: Claire Ortmann de Percin Northumberland / Mariya Licheva / Rahel Dabrowski / Rubén Gómez-Sánchez / Sabrina Berkamp / Philipp Schönnenbeck / Martin Graef / Claudine Kraft / Carsten Sachse /
Abstract: The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid ...The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features reveal surrounding and membrane-attached vesicles. The dimensions of the enlarged rims are consistent with fusion of cytosolic vesicles with the growing phagophore. High-resolution imaging in this study suggests that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.
History
DepositionApr 29, 2026-
Header (metadata) releaseOct 7, 2026-
Map releaseOct 7, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_57826.png

Downloads & links

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Map

FileDownload / File: emd_57826.map.gz / Format: CCP4 / Size: 2.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationphagophore in direct contact with the phospholipid monolayer at the surface of a lipid droplet
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.95 Å/pix.
x 450 pix.
= 6277.5 Å		13.95 Å/pix.
x 1022 pix.
= 14256.899 Å		13.95 Å/pix.
x 1440 pix.
= 20088. 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: 13.95 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.0025003979 - 0.0012592725
Average (Standard dev.)-0.00000021658174 (±0.00010170702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10221440450
Spacing14401022450
CellA: 20088.0 Å / B: 14256.899 Å / C: 6277.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed ...

EntireName: Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed under native promoters from plasmids while the native gene copies were knocked out
Components
  • Cell: Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed under native promoters from plasmids while the native gene copies were knocked out

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Supramolecule #1: Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed ...

SupramoleculeName: Atg2-PM4-3xmScarlet mutant Atg9-3xmTagBFP2 mutant both expressed under native promoters from plasmids while the native gene copies were knocked out
type: cell / ID: 1 / Parent: 0
Details: Atg2-PM4 mutant protein previously generated and characterised (doi:10.1083/jcb.201710116)
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast)

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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 R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: SILICON DIOXIDE / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 80 % / Chamber temperature: 303 K / Instrument: FEI VITROBOT MARK IV
Cryo protectantnone
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.01 / Focused ion beam - Duration: 10 / Focused ion beam - Temperature: 77 K / Focused ion beam - Initial thickness: 600 / Focused ion beam - Final thickness: 280
Focused ion beam - Details: polishing was done manually so duration is an approximation. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values ...Focused ion beam - Details: polishing was done manually so duration is an approximation. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.
Fiducial markerManufacturer: Thermo Fisher Scientific / Diameter: 100 nm

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.16 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 24000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: AreTomo (ver. 1) / Number images used: 60
CTF correctionSoftware - Name: Warp / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION

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