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- EMDB-56238: In situ cryo-ET subtomogram averaged map of Flotillin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-56238
TitleIn situ cryo-ET subtomogram averaged map of Flotillin complex
Map dataFlotillin sub-tomogram averaged post-processed map
Sample
  • Cell: Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET
KeywordsComplex / Flotillin / Lysosome / Endosome / Endo-membrane protein / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 18.74 Å
AuthorsLi D / Lizarrondo J / Wilfling F
Funding support United States, 1 items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP) 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 7, 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_56238.png

Downloads & links

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Map

FileDownload / File: emd_56238.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFlotillin sub-tomogram averaged post-processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.18 Å/pix.
x 224 pix.
= 487.424 Å		2.18 Å/pix.
x 224 pix.
= 487.424 Å		2.18 Å/pix.
x 224 pix.
= 487.424 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.176 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.146
Minimum - Maximum-0.1510892 - 0.3613496
Average (Standard dev.)0.018708393 (±0.06504973)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 487.424 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_56238_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Flotillin sub-tomogram averaged unfiltered halfmap 1

Fileemd_56238_half_map_1.map
AnnotationFlotillin sub-tomogram averaged unfiltered halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Flotillin sub-tomogram averaged unfiltered halfmap 2

Fileemd_56238_half_map_2.map
AnnotationFlotillin sub-tomogram averaged unfiltered halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET

EntireName: Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET
Components
  • Cell: Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET

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Supramolecule #1: Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET

SupramoleculeName: Flotillin complex in HeLa TMEM192-3xHA cells using in situ cryo-ET
type: cell / ID: 1 / Parent: 0
Details: incubated overnight Dextran647 for correlative targeting
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.5
Details: Dulbecco's Modified Eagle Medium (DMEM) - high glucose With sodium bicarbonate, L-glutamine and sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
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
Details: The grid was coated using laminin (Sigma-Aldrich, L4544) for 1h before cell seeding
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 310 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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 / Nominal magnification: 42000
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 reconstructionApplied symmetry - Point group: C22 (22 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 18.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Software - details: 3.1.4 / Number subtomograms used: 213
ExtractionNumber tomograms: 85 / Number images used: 1176 / Method: Template Matching / Software - Name: Warp / Software - details: 1.0.9
Details: TM using GapstopTM using previously generated map from manual picking
CTF correctionSoftware - Name: Warp / Software - details: 1.0.9 / Type: PHASE FLIPPING ONLY
Final angle assignmentType: OTHER / Software - Name: RELION / Software - details: 3.1.4
FSC plot (resolution estimation)

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