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- EMDB-23756: Tomogram of conditioned medium generated from PC12 14A2.6 cells s... -

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Basic information

Entry
Database: EMDB / ID: EMD-23756
TitleTomogram of conditioned medium generated from PC12 14A2.6 cells showing mHTT assemblies
Map dataTomogram of conditioned medium generated from PC12 14A2.6 cells showing mHTT assemblies
Sample
  • Organelle or cellular component: Mutant huntingtin exon 1 fused with enhanced green fluorescent protein in conditioned medium collected from induced PC12 14A2.6 cells (EV-enclosed)
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsNunn K / Jiang J / Dai W
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MB-2046180 United States
CitationJournal: Biochem Biophys Res Commun / Year: 2021
Title: Structural insight into transmissive mutant huntingtin species by correlative light and electron microscopy and cryo-electron tomography.
Authors: Xuyuan Kuang / Kyle Nunn / Jennifer Jiang / Paul Castellano / Uttara Hardikar / Arianna Horgan / Joyce Kong / Zhiqun Tan / Wei Dai /
Abstract: Aggregates of mutant huntingtin (mHTT) containing an expanded polyglutamine (polyQ) tract are hallmarks of Huntington's Disease (HD). Studies have shown that mHTT can spread between cells, leading to ...Aggregates of mutant huntingtin (mHTT) containing an expanded polyglutamine (polyQ) tract are hallmarks of Huntington's Disease (HD). Studies have shown that mHTT can spread between cells, leading to the propagation of misfolded protein pathology. However, the structure of transmissive mHTT species, and the molecular mechanisms underlying their transmission remain unknown. Using correlative light and electron microscopy (CLEM) and cryo-electron tomography (cryo-ET), we identified two types of aggregation-prone granules in conditioned medium from PC12 cells expressing a mHTT N-terminal fragment: densities enclosed by extracellular vesicles (EVs), and uncoated, amorphous meshworks of heterogeneous oligomers that co-localize with clusters of EVs. In vitro assays confirmed that liposomes induce condensation of polyQ oligomers into higher-order assemblies, resembling the uncoated meshworks observed in PC12 conditioned medium. Our findings provide novel insights into formation and architecture of transmissive mHTT proteins, and highlight the potential role of EVs as both carriers and modulators of transmissive mHTT proteins.
History
DepositionApr 2, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateNov 9, 2022-
Current statusNov 9, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_23756.png

Downloads & links

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Map

FileDownload / File: emd_23756.map.gz / Format: CCP4 / Size: 556.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of conditioned medium generated from PC12 14A2.6 cells showing mHTT assemblies
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.2 Å/pix.
x 166 pix.
= 2855.2 Å		17.2 Å/pix.
x 954 pix.
= 16408.801 Å		17.2 Å/pix.
x 921 pix.
= 15841.201 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: 17.2 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-11.925793 - 22.364084
Average (Standard dev.)-3.5102232e-07 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin107-21-34
Dimensions954921166
Spacing921954166
CellA: 15841.201 Å / B: 16408.8 Å / C: 2855.2002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Mutant huntingtin exon 1 fused with enhanced green fluorescent pr...

EntireName: Mutant huntingtin exon 1 fused with enhanced green fluorescent protein in conditioned medium collected from induced PC12 14A2.6 cells (EV-enclosed)
Components
  • Organelle or cellular component: Mutant huntingtin exon 1 fused with enhanced green fluorescent protein in conditioned medium collected from induced PC12 14A2.6 cells (EV-enclosed)

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Supramolecule #1: Mutant huntingtin exon 1 fused with enhanced green fluorescent pr...

SupramoleculeName: Mutant huntingtin exon 1 fused with enhanced green fluorescent protein in conditioned medium collected from induced PC12 14A2.6 cells (EV-enclosed)
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Details: Densities enclosed by extracellular vesicles
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridPretreatment - Type: GLOW DISCHARGE / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293.15 K / Instrument: LEICA EM GP
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 31000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 40

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