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- PDB-2n1f: Structure and assembly of the mouse ASC filament by combined NMR ... -

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Basic information

Entry
Database: PDB / ID: 2n1f
TitleStructure and assembly of the mouse ASC filament by combined NMR spectroscopy and cryo-electron microscopy
ComponentsApoptosis-associated speck-like protein
KeywordsAPOPTOSIS / mouse ASC filament / ASC Apoptosis-associated speck like protein containing a CARD / PYRIN domain / inflammasomes / death domain
Function / homology
Function and homology information


CLEC7A/inflammasome pathway / The NLRP3 inflammasome / Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / 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 / IkappaB kinase complex ...CLEC7A/inflammasome pathway / The NLRP3 inflammasome / Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / 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 / IkappaB kinase complex / AIM2 inflammasome complex / macropinocytosis / NLRP1 inflammasome complex / canonical inflammasome complex / interleukin-6 receptor binding / positive regulation of adaptive immune response / NLRP3 inflammasome complex assembly / BMP receptor binding / 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 / positive regulation of actin filament polymerization / tropomyosin binding / pyroptotic inflammatory response / positive regulation of activated T cell proliferation / positive regulation of release of cytochrome c from mitochondria / 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 phagocytosis / positive regulation of chemokine production / tumor necrosis factor-mediated signaling pathway / negative regulation of canonical NF-kappaB signal transduction / positive regulation of defense response to virus by host / Neutrophil degranulation / negative regulation of cytokine production involved in inflammatory response / activation of innate immune response / positive regulation of interleukin-1 beta production / positive regulation of interleukin-8 production / response to bacterium / positive regulation of JNK cascade / positive regulation of non-canonical NF-kappaB signal transduction / regulation of protein stability / protein homooligomerization / 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 / protease binding / regulation of inflammatory response / defense response to Gram-negative bacterium / regulation of apoptotic process / defense response to virus / microtubule / transmembrane transporter binding / positive regulation of canonical NF-kappaB signal transduction / positive regulation of ERK1 and ERK2 cascade / defense response to Gram-positive bacterium / protein dimerization activity / 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 / cytosol / cytoplasm
Similarity search - Function
CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / Death Domain, Fas / PAAD/DAPIN/Pyrin domain / Death Domain, Fas / CARD domain / CARD caspase recruitment domain profile. ...CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / Death Domain, Fas / PAAD/DAPIN/Pyrin domain / Death Domain, Fas / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Apoptosis-associated speck-like protein containing a CARD
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodSOLID-STATE NMR / ELECTRON MICROSCOPY / helical reconstruction / simulated annealing / cryo EM / Resolution: 4 Å
Model detailslowest energy, model1
AuthorsSborgi, 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.
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
DepositionApr 1, 2015Deposition site: BMRB / Processing site: RCSB
Revision 1.0Oct 14, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 28, 2015Group: Database references
Revision 1.2Nov 11, 2015Group: Database references
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id
Revision 1.4Jun 14, 2023Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status / pdbx_nmr_spectrometer
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_cs / _pdbx_database_status.status_code_mr / _pdbx_database_status.status_code_nmr_data / _pdbx_nmr_spectrometer.model
Revision 1.5May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Assembly

Deposited unit
A: Apoptosis-associated speck-like protein
B: Apoptosis-associated speck-like protein
C: Apoptosis-associated speck-like protein
D: Apoptosis-associated speck-like protein
E: Apoptosis-associated speck-like protein
F: Apoptosis-associated speck-like protein
G: Apoptosis-associated speck-like protein
H: Apoptosis-associated speck-like protein
I: Apoptosis-associated speck-like protein
J: Apoptosis-associated speck-like protein
K: Apoptosis-associated speck-like protein
L: Apoptosis-associated speck-like protein
M: Apoptosis-associated speck-like protein
N: Apoptosis-associated speck-like protein
O: Apoptosis-associated speck-like protein


Theoretical massNumber of molelcules
Total (without water)151,78015
Polymers151,78015
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 100structures with the lowest energy
RepresentativeModel #1lowest energy
SymmetryHelical 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 °)
DetailsThe helical parameters generate the filament from any single chain.

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Components

#1: Protein
Apoptosis-associated speck-like protein / mASC


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

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Experiment

Experiment
Method
SOLID-STATE NMR
ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
111NCACX
121NCOCX
131NCACB
141CANCO
151CCC
161NCA
171NCO
18120msDARR
191N(CO)CACB
1101CAN(CO)CA
1111N(CA)CBCX
1121100msPDSD

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Sample preparation

Component
IDNameTypeParent-ID
1Mouse ASC-PYD filamentCOMPLEX0
2ASC filament1
Buffer solutionName: 25 mM Tris, 300 mM sodium chloride / pH: 8 / Details: 25 mM Tris, 300 mM sodium chloride
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: Grids were blotted for one second before plunging into liquid ethane (FEI VITROBOT MARK IV).
DetailsContents: 15 mg [U-100% 13C; U-100% 15N] ASC filament, 90% H2O/10% D2O
Solvent system: 90% H2O/10% D2O
SampleConc.: 15 mg/mL / Component: ASC filament-1 / Isotopic labeling: [U-100% 13C; U-100% 15N]
Sample conditionsPressure: ambient / Temperature: 288 K

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 10, 2014
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DIFFRACTION / Nominal magnification: 22500 X
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle max: 12 ° / Tilt angle min: -12 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: DIRECT ELECTRON DE-10 (5k x 4k)
Image scansNum. digital images: 21138
NMR spectrometerType: Bruker Avance / Manufacturer: Bruker / Model: AVANCE / Field strength: 850 MHz

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Processing

EM software
IDNameVersionCategory
1EMAN23D reconstruction
2IHRSR3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: CTFFIND3
Helical symmertyAngular rotation/subunit: 53 ° / Axial rise/subunit: 14.2 Å / Axial symmetry: C3
3D reconstructionMethod: 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 stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms10590 0 0 0 10590
NMR software
NameDeveloperClassification
CYANAGuntert, Mumenthaler and Wuthrichchemical shift assignment
CYANAGuntert, Mumenthaler and Wuthrichdata analysis
CYANAGuntert, Mumenthaler and Wuthrichstructure solution
XPLOR-NIHSchwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 4
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 10

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