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- EMDB-2971: Structure and assembly of the mouse ASC filament -

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Basic information

Entry
Database: EMDB / ID: EMD-2971
TitleStructure and assembly of the mouse ASC filament
Map dataReconstruction of ASC-PYD filament
Sample
  • Sample: Mouse ASC-PYD filament
  • Protein or peptide: mouse ASC filament
KeywordsASC Apoptosis associated speck like protein containing a CARD / CARD caspase recruitment and activation domain / PYD PYRIN domain / PYHIN pyrin domain and hematopoietic expression / interferon inducibility / nuclear localization domain containing / NLR NOD like receptor / BIR Baculovirus IAP repeat domain
Function / homology
Function and homology information


CLEC7A/inflammasome pathway / The NLRP3 inflammasome / NLRP6 inflammasome complex / myosin I binding / Pyrin domain binding / myeloid dendritic cell activation involved in immune response / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / peptidase activator activity involved in apoptotic process / regulation of intrinsic apoptotic signaling pathway / IkappaB kinase complex ...CLEC7A/inflammasome pathway / The NLRP3 inflammasome / NLRP6 inflammasome complex / myosin I binding / Pyrin domain binding / myeloid dendritic cell activation involved in immune response / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / peptidase activator activity involved in apoptotic process / regulation of intrinsic apoptotic signaling pathway / IkappaB kinase complex / AIM2 inflammasome complex / NLRP1 inflammasome complex / macropinocytosis / canonical inflammasome complex / interleukin-6 receptor binding / BMP receptor binding / NLRP3 inflammasome complex assembly / positive regulation of adaptive immune response / NLRP3 inflammasome complex / negative regulation of interferon-beta production / osmosensory signaling pathway / regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of extrinsic apoptotic signaling pathway / pattern recognition receptor signaling pathway / regulation of GTPase activity / positive regulation of macrophage cytokine production / tropomyosin binding / pyroptotic inflammatory response / positive regulation of actin filament polymerization / positive regulation of release of cytochrome c from mitochondria / positive regulation of activated T cell proliferation / positive regulation of interleukin-10 production / cellular response to interleukin-1 / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / positive regulation of T cell migration / positive regulation of chemokine production / positive regulation of defense response to virus by host / Neutrophil degranulation / negative regulation of canonical NF-kappaB signal transduction / negative regulation of cytokine production involved in inflammatory response / activation of innate immune response / positive regulation of phagocytosis / positive regulation of interleukin-1 beta production / tumor necrosis factor-mediated signaling pathway / positive regulation of interleukin-8 production / positive regulation of JNK cascade / response to bacterium / positive regulation of non-canonical NF-kappaB signal transduction / protein homooligomerization / regulation of protein stability / positive regulation of interleukin-6 production / positive regulation of type II interferon production / positive regulation of inflammatory response / positive regulation of tumor necrosis factor production / cellular response to lipopolysaccharide / regulation of inflammatory response / protease binding / regulation of apoptotic process / defense response to Gram-negative bacterium / defense response to virus / microtubule / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / positive regulation of canonical NF-kappaB signal transduction / protein dimerization activity / defense response to Gram-positive bacterium / regulation of autophagy / inflammatory response / Golgi membrane / innate immune response / neuronal cell body / apoptotic process / nucleolus / endoplasmic reticulum / protein homodimerization activity / mitochondrion / extracellular region / nucleoplasm / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Apoptosis-associated speck-like protein containing a CARD
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodhelical reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsSborgi L / Ravotti F / Dandey VP / Dick MS / Mazur A / Reckel S / Chami M / Scherer S / Bockmann A / Egelman EH ...Sborgi L / Ravotti F / Dandey VP / Dick MS / Mazur A / Reckel S / Chami M / Scherer S / Bockmann A / Egelman EH / Stahlberg H / Broz P / Meier BH / Hiller S
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy.
Authors: Lorenzo Sborgi / Francesco Ravotti / Venkata P Dandey / Mathias S Dick / Adam Mazur / Sina Reckel / Mohamed Chami / Sebastian Scherer / Matthias Huber / Anja Böckmann / Edward H Egelman / ...Authors: Lorenzo Sborgi / Francesco Ravotti / Venkata P Dandey / Mathias S Dick / Adam Mazur / Sina Reckel / Mohamed Chami / Sebastian Scherer / Matthias Huber / Anja Böckmann / Edward H Egelman / Henning Stahlberg / Petr Broz / Beat H Meier / Sebastian Hiller /
Abstract: Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous ...Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.
History
DepositionMar 31, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseOct 14, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2n1f
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2n1f
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2n1f
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2971.jpg

Downloads & links

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Map

FileDownload / File: emd_2971.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of ASC-PYD filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 160 pix.
= 107.2 Å		0.67 Å/pix.
x 200 pix.
= 134. Å		0.67 Å/pix.
x 200 pix.
= 134. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.67 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 20.0 / Movie #1: 25
Minimum - Maximum-47.816951750000001 - 81.899246219999995
Average (Standard dev.)-2.8232131 (±11.493054389999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-79
Dimensions200200160
Spacing200200160
CellA: 134.0 Å / B: 134.0 Å / C: 107.200005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.670.670.67
M x/y/z200200160
origin x/y/z0.0000.0000.000
length x/y/z134.000134.000107.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-79
NC/NR/NS200200160
D min/max/mean-47.81781.899-2.823

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Supplemental data

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Sample components

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Entire : Mouse ASC-PYD filament

EntireName: Mouse ASC-PYD filament
Components
  • Sample: Mouse ASC-PYD filament
  • Protein or peptide: mouse ASC filament

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Supramolecule #1000: Mouse ASC-PYD filament

SupramoleculeName: Mouse ASC-PYD filament / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: mouse ASC filament

MacromoleculeName: mouse ASC filament / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLysS / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8 / Details: 25mM Tris 300mM NaCl
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: vitrified by plunging into liquid nitrogen-cooled liquid ethane
Method: The grids were blotted for 1 s before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateOct 10, 2014
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-10 (5k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 21138 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DIFFRACTION / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle min: -12 / Tilt angle max: 12
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were aligned using IHRSR
Final reconstructionApplied symmetry - Helical parameters - Δz: 14.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 53 °
Applied symmetry - Helical parameters - Axial symmetry: C3 (3 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: EMAN2, IHRSR, SPIDER
CTF correctionDetails: CTFFIND3

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