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- EMDB-55210: In situ cryo-ET tomogram of HeLa TMEM192-3xHA Control cell showca... -

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Basic information

Entry
Database: EMDB / ID: EMD-55210
TitleIn situ cryo-ET tomogram of HeLa TMEM192-3xHA Control cell showcasing an endolysosomal structure.
Map dataIn situ cryo-ET tomogram of HeLa TMEM192-3xHA Control cell.
Sample
  • Cell: HeLa TMEM192-3xHA
KeywordsLysosome / Endosome / Ultrastructure / Organelle / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsKraus F / Li D / Wilfling F / Harper JW
Funding support United States, 1 items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000282 and 024268 United States
CitationJournal: bioRxiv / Year: 2025
Title: Proteome Landscapes Decode Organelle Vulnerabilities in cortical and dopaminergic-like induced neurons Across Lysosomal Storage Disorders.
Authors: Felix Kraus / Yuchen He / Yizhi Jiang / Delong Li / Yohannes A Ambaw / Federico M Gasparoli / Joao A Paulo / Tobias C Walther / Robert Farese / Steven P Gygi / Florian Wilfling / J Wade Harper
Abstract: Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy and recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) ...Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy and recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) comprise a broad group of diseases affecting lysosomal degradation, ion flux, and lipid catabolism. Within this group, sphingolipidoses genes involved in glycosphingolipid breakdown are known (GBA1) or candidate (SMPD1, ASAH1) risk factors for Parkinsons Disease, though disease mechanisms remain unclear. Using our previously reported LSD mutant proteomic landscape in HeLa cells, we observed pronounced variability in endolysosomal proteome signatures among sphingolipid pathway mutants, with ASAH1 knockout cells showing altered lysosomal lipid composition, impaired endocytic trafficking, and disrupted ultrastructure by cryo-electron tomography. To extend these findings in a more physiologic context, we generated a human embryonic stem (ES) cell library comprising 23 LSD gene knockouts and profiled proteomic changes during differentiation into cortical and midbrain dopaminergic neurons over a 7 to 10 week period. LSD mutants exhibited lineage-specific alterations in organellar proteomes, revealing diverse vulnerabilities. Notably, GBA1 knockout and ASAH1knockout dopaminergic neurons showed disruptions in synaptic and mitochondrial compartments, correlating with impaired dopaminergic neuronal firing and disrupted presynaptic protein localization. This LSD mutant toolkit and associated proteomic landscape provides a resource for defining molecular signatures of LSD gene loss and highlights convergence of lysosomal dysfunction, synaptic integrity, and mitochondrial health as potential links between sphingolipidoses and PD risk.
History
DepositionSep 26, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 3, 2025-
Current statusDec 3, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55210.png

Downloads & links

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Map

FileDownload / File: emd_55210.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ cryo-ET tomogram of HeLa TMEM192-3xHA Control cell.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.88 Å/pix.
x 574 pix.
= 4525.416 Å		7.88 Å/pix.
x 1024 pix.
= 8073.216 Å		7.88 Å/pix.
x 1024 pix.
= 8073.216 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: 7.884 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.046244163 - 0.018145997
Average (Standard dev.)-0.000033939763 (±0.00057170686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10241024574
Spacing10241024574
CellA: 8073.216 Å / B: 8073.216 Å / C: 4525.416 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

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

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Supramolecule #1: HeLa TMEM192-3xHA

SupramoleculeName: HeLa TMEM192-3xHA / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa TMEM192-3xHA Control

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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: 1 / 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 Aquilos2 FIB/SEM. 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

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 2.4 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: 3.0 µm / Nominal magnification: 64000
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. 4.12.62) / Software - details: AreTomo2 v.1.0.0 / Number images used: 61
CTF correctionType: PHASE FLIPPING ONLY

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