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- EMDB-56327: In situ cryo-ET tomogram of lysosomal structure in untreated rat ... -

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Basic information

Entry
Database: EMDB / ID: EMD-56327
TitleIn situ cryo-ET tomogram of lysosomal structure in untreated rat hippocampal neurons
Map dataIn situ cryo-ET tomogram of lysosomal structure in untreated rat hippocampal neuron.
Sample
  • Cell: Rat hippocampal neuron
KeywordsLysosome / Endolysosomal system / neuron / rat / ENDOCYTOSIS
Biological speciesRattus (rats)
Methodelectron tomography / cryo EM
AuthorsLi D / Schwarz A / 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 12, 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_56327.png

Downloads & links

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Map

FileDownload / File: emd_56327.map.gz / Format: CCP4 / Size: 2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ cryo-ET tomogram of lysosomal structure in untreated rat hippocampal neuron.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
12.15 Å/pix.
x 500 pix.
= 6075. Å		12.15 Å/pix.
x 1024 pix.
= 12441.6 Å		12.15 Å/pix.
x 1024 pix.
= 12441.6 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: 12.15 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-22.734781000000002 - 3.9441652
Average (Standard dev.)0.0018646427 (±0.26879454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10241024500
Spacing10241024500
CellA: 12441.6 Å / B: 12441.6 Å / C: 6075.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Rat hippocampal neuron

EntireName: Rat hippocampal neuron
Components
  • Cell: Rat hippocampal neuron

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Supramolecule #1: Rat hippocampal neuron

SupramoleculeName: Rat hippocampal neuron / type: cell / ID: 1 / Parent: 0
Details: rat hippocampal neurons were prepared from P0 or P1 rat pups (Sprague Dawley, IGS, Crl:CD(SD), Charles River Laboratories, RRID:RGD 734476)
Source (natural)Organism: Rattus (rats) / Organ: Brain / Tissue: Hippocampus

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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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 310 K / Instrument: LEICA EM GP
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: The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2 FIB/SM. 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

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k 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 / Nominal defocus max: 7.0 µm / Nominal defocus min: 4.0 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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.12) / Number images used: 60
CTF correctionType: PHASE FLIPPING ONLY

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