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Yorodumi- EMDB-13586: Cryo-electron tomography of ASC signalling sites in pyroptotic ce... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13586 | |||||||||
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Title | Cryo-electron tomography of ASC signalling sites in pyroptotic cells (2) | |||||||||
Map data | Cryo-ET of ASC-mCerulean speck in iBMDMs | |||||||||
Sample |
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Keywords | ASC-mCerulean speck / PROTEIN FIBRIL | |||||||||
Biological species | Mus musculus (house mouse) | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Liu 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
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_13586.map.gz | 416 MB | EMDB map data format | |
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Header (meta data) | emd-13586-v30.xml emd-13586.xml | 10.4 KB 10.4 KB | Display Display | EMDB header |
Images | emd_13586.png | 237.8 KB | ||
Filedesc metadata | emd-13586.cif.gz | 4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13586 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13586 | HTTPS FTP |
-Validation report
Summary document | emd_13586_validation.pdf.gz | 427.5 KB | Display | EMDB validaton report |
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Full document | emd_13586_full_validation.pdf.gz | 427.1 KB | Display | |
Data in XML | emd_13586_validation.xml.gz | 5.1 KB | Display | |
Data in CIF | emd_13586_validation.cif.gz | 5.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13586 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13586 | HTTPS FTP |
-Related structure data
Related structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13586.map.gz / Format: CCP4 / Size: 450 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||
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Annotation | Cryo-ET of ASC-mCerulean speck in iBMDMs | ||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 13.58 Å | ||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : FIB milled iBMDMs
Entire | Name: FIB milled iBMDMs |
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Components |
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-Supramolecule #1: FIB milled iBMDMs
Supramolecule | Name: FIB milled iBMDMs / type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Mus musculus (house mouse) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 Details: DMEM medium supplemented with 10% heat-inactivated FBS (Gibco) |
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Grid | Model: 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 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: LEICA EM GP |
Sectioning | Focused 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.. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number real images: 121 / Average electron dose: 1.3 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 7.0 µm / Nominal defocus min: 4.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Resolution method: OTHER / Software - Name: eTomo / Number images used: 99 |
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