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- EMDB-56297: In situ cryo-ET of lysosome damaged by LLOMe (0.5mM, 60min) encap... -

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Entry
Database: EMDB / ID: EMD-56297
TitleIn situ cryo-ET of lysosome damaged by LLOMe (0.5mM, 60min) encapsulated in an autophagosome in HeLa TMEM192-3xHA cell.
Map dataIn situ cryo-ET of lysosome damaged by LLOMe (0.5mM, 60min) encapsulated in an autophagosome in HeLa TMEM192-3xHA cell.
Sample
  • Cell: HeLa TMEM192-3xHA cell
KeywordsLysosome / HeLa cell / Endolysomal system / organelle / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsLi D / Wilfling F
Funding support United States, 1 items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000282/ASAP-024268 United States
CitationJournal: bioRxiv / Year: 2026
Title: Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes.
Authors: Delong Li / Wenxin Zhang / Michaela Medina / Jan F M Stuke / Andre Schwarz / Jonas Brill / Johann Brenner / Felix Kraus / Simon Ohlerich / Javier Lizarrondo / Jeremy Pflaum / Julia H Grass / ...Authors: Delong Li / Wenxin Zhang / Michaela Medina / Jan F M Stuke / Andre Schwarz / Jonas Brill / Johann Brenner / Felix Kraus / Simon Ohlerich / Javier Lizarrondo / Jeremy Pflaum / Julia H Grass / Lena-Marie Soltow / Dietmar Hammerschmid / Natalie Weber / Sonja Welsch / Julian D Langer / Maike Windbergs / J Wade Harper / Erin Schuman / Gerhard Hummer / Danielle A Grotjahn / Florian Wilfling
Abstract: Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester ...Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.
History
DepositionJan 11, 2026-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateFeb 18, 2026-
Current statusFeb 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56297.png

Downloads & links

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Map

FileDownload / File: emd_56297.map.gz / Format: CCP4 / Size: 2.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ cryo-ET of lysosome damaged by LLOMe (0.5mM, 60min) encapsulated in an autophagosome in HeLa TMEM192-3xHA cell.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.7 Å/pix.
x 524 pix.
= 4560.896 Å		8.7 Å/pix.
x 1440 pix.
= 12533.761 Å		8.7 Å/pix.
x 1022 pix.
= 8895.488 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: 8.704 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-8.748570000000001 - 2.2851129
Average (Standard dev.)-0.0022004936 (±0.093989804)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401022524
Spacing10221440524
CellA: 8895.488 Å / B: 12533.761 Å / C: 4560.8965 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : HeLa TMEM192-3xHA cell

EntireName: HeLa TMEM192-3xHA cell
Components
  • Cell: HeLa TMEM192-3xHA cell

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Supramolecule #1: HeLa TMEM192-3xHA cell

SupramoleculeName: HeLa TMEM192-3xHA cell / type: cell / ID: 1 / Parent: 0
Details: treated with LLOMe (Cayman Chemicals, 16008) at 0.5mM for 60min.
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa

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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: 7.4
GridModel: Quantifoil R1/4 / Material: GOLD / Mesh: 200 / Support film - Material: SILICON DIOXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 310 K / Instrument: LEICA EM GP
Detailstreated with LLOMe (Cayman Chemicals, 16008) at 0.5mM for 60min.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.5 / Focused ion beam - Duration: 120 / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 130
Focused ion beam - Details: using AutoTEM. The value given for _em_focused_ion_beam.instrument is Aquilos 2 FIB/SEM. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
Fiducial markerManufacturer: Dynabeads Thermo Fisher Scientific, acid65011 / Diameter: 1 nm

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen 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 reconstructionSoftware:
Namedetails
IMOD (ver. 4.12.62)
AreTomo (ver. 1.0.0)AreTomo2, Denoising done in Isonet2

Number images used: 61
CTF correctionSoftware - Name: Gctf / Type: PHASE FLIPPING ONLY

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