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- EMDB-51701: In situ cryo-electron tomogram of a multi-lamellar vesicle in a N... -

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Basic information

Entry
Database: EMDB / ID: EMD-51701
TitleIn situ cryo-electron tomogram of a multi-lamellar vesicle in a NPC2-/- HeLa cell. #1
Map dataHighly organized membrane structures with NPC2-/- MLVs
Sample
  • Cell: HeLa TMEM192-3xHA NPC2-/-
KeywordsLysosomal storage disorders / autophagy / Multi-lamellar vesicle / cholesterol export / endocytosis / cargo delivery / LIPID TRANSPORT
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsKraus F / He Y / Swarup S / Overmyer KA / Jiang Y / Brenner J / Capitanio C / Bieber A / Jen A / Nightingale NM ...Kraus F / He Y / Swarup S / Overmyer KA / Jiang Y / Brenner J / Capitanio C / Bieber A / Jen A / Nightingale NM / Anderson BJ / Lee C / Paulo JA / Smith IR / Plitzko JM / Gygi SP / Schulman BA / Wilfling F / Coon JJ / Harper JW
Funding support United States, 1 items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000282 United States
CitationJournal: Sci Adv / Year: 2025
Title: Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.
Authors: Felix Kraus / Yuchen He / Sharan Swarup / Katherine A Overmyer / Yizhi Jiang / Johann Brenner / Cristina Capitanio / Anna Bieber / Annie Jen / Nicole M Nightingale / Benton J Anderson / Chan ...Authors: Felix Kraus / Yuchen He / Sharan Swarup / Katherine A Overmyer / Yizhi Jiang / Johann Brenner / Cristina Capitanio / Anna Bieber / Annie Jen / Nicole M Nightingale / Benton J Anderson / Chan Lee / Joao A Paulo / Ian R Smith / Jürgen M Plitzko / Steven P Gygi / Brenda A Schulman / Florian Wilfling / Joshua J Coon / J Wade Harper /
Abstract: Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is ...Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in and mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.
History
DepositionOct 2, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51701.png

Downloads & links

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Map

FileDownload / File: emd_51701.map.gz / Format: CCP4 / Size: 564 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHighly organized membrane structures with NPC2-/- MLVs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
11.72 Å/pix.
x 141 pix.
= 1652.52 Å		11.72 Å/pix.
x 1024 pix.
= 12001.28 Å		11.72 Å/pix.
x 1024 pix.
= 12001.28 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: 11.72 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-327.606049999999982 - 174.683600000000013
Average (Standard dev.)1.1199864 (±6.75972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin2010
Dimensions10241024141
Spacing10241024141
CellA: 12001.28 Å / B: 12001.28 Å / C: 1652.52 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 NPC2-/-

EntireName: HeLa TMEM192-3xHA NPC2-/-
Components
  • Cell: HeLa TMEM192-3xHA NPC2-/-

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Supramolecule #1: HeLa TMEM192-3xHA NPC2-/-

SupramoleculeName: HeLa TMEM192-3xHA NPC2-/- / type: cell / ID: 1 / Parent: 0
Details: Modified with CRISPR/CAS9 with target sites determined using CHOPCHOP.
Source (natural)Organism: Homo sapiens (human)

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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 R1/4 / Material: GOLD / Mesh: 200 / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsHeLa (TMEM192-HA) NPC2-/- cells were cultured on Poly-L-Lysine coated gold grids over night. The next day, cells were starved for 6h in EBSS. 10% glycerol was added shortly before plunging.
Cryo protectant10% glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 3 / 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 Thermo Fisher Arctis PFIB. 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: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.5 µm / Nominal defocus min: 3.5 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 reconstructionAlgorithm: BACK PROJECTION / Software - Version: 1.3 / Software - details: Aretomo / Number images used: 47

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