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- EMDB-13586: Cryo-electron tomography of ASC signalling sites in pyroptotic ce... -

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Basic information

Entry
Database: EMDB / ID: EMD-13586
TitleCryo-electron tomography of ASC signalling sites in pyroptotic cells (2)
Map dataCryo-ET of ASC-mCerulean speck in iBMDMs
Sample
  • Cell: FIB milled iBMDMs
KeywordsASC-mCerulean speck / PROTEIN FIBRIL
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsLiu Y / Modis Y / Alemayehu H / Hopkins LJ / Borgeaud AC / Heroven C / Howe JD / Boulanger J / Bryant CE / Zhai H
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.
Authors: Yangci Liu / Haoming Zhai / Helen Alemayehu / Jérôme Boulanger / Lee J Hopkins / Alicia C Borgeaud / Christina Heroven / Jonathan D Howe / Kendra E Leigh / Clare E Bryant / Yorgo Modis /
Abstract: NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, ...NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.
History
DepositionSep 16, 2021-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13586.png

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Map

FileDownload / File: emd_13586.map.gz / Format: CCP4 / Size: 450 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-ET of ASC-mCerulean speck in iBMDMs
Voxel sizeX=Y=Z: 13.58 Å
Density
Minimum - Maximum-114.357119999999995 - 93.054665
Average (Standard dev.)0.0029086974 (±16.782791)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin007
Dimensions1440102480
Spacing1024144080
CellA: 13905.92 Å / B: 19555.2 Å / C: 1086.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : FIB milled iBMDMs

EntireName: FIB milled iBMDMs
Components
  • Cell: FIB milled iBMDMs

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Supramolecule #1: FIB milled iBMDMs

SupramoleculeName: FIB milled iBMDMs / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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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
Details: DMEM medium supplemented with 10% heat-inactivated FBS (Gibco)
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 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: 1500 / Focused ion beam - Temperature: 83 K / Focused ion beam - Initial thickness: 10000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: Lamella were made using a Scios DualBeam FIB/SEM (FEI) equipped with a Quorum cryo-stage (PP3010T). Rough milling was performed using 0.5 to 0.1 nA current. Fine milling ...Focused ion beam - Details: Lamella were made using a Scios DualBeam FIB/SEM (FEI) equipped with a Quorum cryo-stage (PP3010T). Rough milling was performed using 0.5 to 0.1 nA current. Fine milling was done using 50 to 10 pA current..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.0 µm / Nominal defocus min: 4.0 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 121 / Average electron dose: 1.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution method: OTHER / Software - Name: eTomo / Number images used: 99

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