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Yorodumi- PDB-2n1f: Structure and assembly of the mouse ASC filament by combined NMR ... -
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-Basic information
Entry | Database: PDB / ID: 2n1f | ||||||
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Title | Structure and assembly of the mouse ASC filament by combined NMR spectroscopy and cryo-electron microscopy | ||||||
Components | Apoptosis-associated speck-like protein | ||||||
Keywords | APOPTOSIS / mouse ASC filament / ASC Apoptosis-associated speck like protein containing a CARD / PYRIN domain / inflammasomes / death domain | ||||||
Function / homology | Function and homology information interleukin-1 beta production / CLEC7A/inflammasome pathway / positive regulation of chemokine production => GO:0032722 / Pyrin domain binding / myosin I binding / regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / peptidase activator activity involved in apoptotic process ...interleukin-1 beta production / CLEC7A/inflammasome pathway / positive regulation of chemokine production => GO:0032722 / Pyrin domain binding / myosin I binding / regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / peptidase activator activity involved in apoptotic process / myeloid dendritic cell activation / IkappaB kinase complex / AIM2 inflammasome complex / macropinocytosis / interleukin-6 receptor binding / NLRP1 inflammasome complex / positive regulation of adaptive immune response / NLRP3 inflammasome complex / BMP receptor binding / negative regulation of protein serine/threonine kinase activity / negative regulation of interferon-beta production / positive regulation of cysteine-type endopeptidase activity / activation of cysteine-type endopeptidase activity / regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of extrinsic apoptotic signaling pathway / cysteine-type endopeptidase activity involved in apoptotic process / negative regulation of NF-kappaB transcription factor activity / regulation of GTPase activity / positive regulation of actin filament polymerization / tropomyosin binding / positive regulation of activated T cell proliferation / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of release of cytochrome c from mitochondria / positive regulation of interleukin-10 production / intrinsic apoptotic signaling pathway by p53 class mediator / cellular response to interleukin-1 / negative regulation of cytokine production involved in inflammatory response / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / positive regulation of T cell migration / negative regulation of canonical NF-kappaB signal transduction / positive regulation of phagocytosis / positive regulation of defense response to virus by host / tumor necrosis factor-mediated signaling pathway / activation of innate immune response / Neutrophil degranulation / positive regulation of interleukin-1 beta production / regulation of autophagy / positive regulation of interleukin-8 production / positive regulation of JNK cascade / response to bacterium / regulation of protein stability / protein homooligomerization / positive regulation of DNA-binding transcription factor activity / positive regulation of interleukin-6 production / positive regulation of type II interferon production / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of tumor necrosis factor production / positive regulation of T cell activation / cellular response to tumor necrosis factor / positive regulation of NF-kappaB transcription factor activity / regulation of inflammatory response / protease binding / cellular response to lipopolysaccharide / regulation of apoptotic process / defense response to virus / defense response to Gram-negative bacterium / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / protein dimerization activity / inflammatory response / positive regulation of apoptotic process / Golgi membrane / innate immune response / neuronal cell body / nucleolus / apoptotic process / enzyme binding / endoplasmic reticulum / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular region / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Mus musculus (house mouse) | ||||||
Method | SOLID-STATE NMR / ELECTRON MICROSCOPY / helical reconstruction / simulated annealing / cryo EM / Resolution: 4 Å | ||||||
Model details | lowest energy, model1 | ||||||
Authors | Sborgi, L. / Ravotti, F. / Dandey, V. / Dick, M. / Mazur, A. / Reckel, S. / Chami, M. / Scherer, S. / Bockmann, A. / Egelman, E. ...Sborgi, L. / Ravotti, F. / Dandey, V. / Dick, M. / Mazur, A. / Reckel, S. / Chami, M. / Scherer, S. / Bockmann, A. / Egelman, E. / Stahlberg, H. / Broz, P. / Meier, B. / Hiller, S. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 2n1f.cif.gz | 2.1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb2n1f.ent.gz | 1.9 MB | Display | PDB format |
PDBx/mmJSON format | 2n1f.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/n1/2n1f ftp://data.pdbj.org/pub/pdb/validation_reports/n1/2n1f | HTTPS FTP |
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-Related structure data
Related structure data | 2971MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
Other databases |
-Links
-Assembly
Deposited unit |
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1 |
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NMR ensembles |
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Symmetry | Helical symmetry: (Circular symmetry: 3 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 8 / Rise per n subunits: 14.2 Å / Rotation per n subunits: 53 °) | |||||||||
Details | The helical parameters generate the filament from any single chain. |
-Components
#1: Protein | Mass: 10118.684 Da / Num. of mol.: 15 / Fragment: pyrin domain (UNP residues 2-90) Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Pycard, Asc / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9EPB4 |
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-Experimental details
-Experiment
Experiment |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction | ||||||||||||||||||||||||||||||||||||||||||||||||||||
NMR experiment |
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-Sample preparation
Component |
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Buffer solution | Name: 25 mM Tris, 300 mM sodium chloride / pH: 8 / Details: 25 mM Tris, 300 mM sodium chloride | ||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE Details: Grids were blotted for one second before plunging into liquid ethane (FEI VITROBOT MARK IV). | ||||||||||||
Details | Contents: 15 mg [U-100% 13C; U-100% 15N] ASC filament, 90% H2O/10% D2O Solvent system: 90% H2O/10% D2O | ||||||||||||
Sample | Conc.: 15 mg/mL / Component: ASC filament-1 / Isotopic labeling: [U-100% 13C; U-100% 15N] | ||||||||||||
Sample conditions | Pressure: ambient / Temperature: 288 K |
-Data collection
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Date: Oct 10, 2014 |
Electron gun | Electron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: DIFFRACTION / Nominal magnification: 22500 X |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle max: 12 ° / Tilt angle min: -12 ° |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: DIRECT ELECTRON DE-10 (5k x 4k) |
Image scans | Num. digital images: 21138 |
NMR spectrometer | Type: Bruker Avance / Manufacturer: Bruker / Model: AVANCE / Field strength: 850 MHz |
-Processing
EM software |
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CTF correction | Details: CTFFIND3 | ||||||||||||||||
Helical symmerty | Angular rotation/subunit: 53 ° / Axial rise/subunit: 14.2 Å / Axial symmetry: C3 | ||||||||||||||||
3D reconstruction | Method: layer line analysis / Resolution: 4 Å / Resolution method: FSC 0.5 CUT-OFF / Nominal pixel size: 0.67 Å / Actual pixel size: 0.67 Å / Symmetry type: HELICAL | ||||||||||||||||
Refinement step | Cycle: LAST
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NMR software |
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Refinement | Method: simulated annealing / Software ordinal: 4 | ||||||||||||||||
NMR representative | Selection criteria: lowest energy | ||||||||||||||||
NMR ensemble | Conformer selection criteria: structures with the lowest energy Conformers calculated total number: 100 / Conformers submitted total number: 10 |