+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2901 | |||||||||
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Title | Cryo electron tomography of Naip5/Nlrc4 inflammasome | |||||||||
Map data | sub tomogram average of NAIP5/NLRC4 polymer | |||||||||
Sample |
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Keywords | NLRs / NAIP5 / NLRC4 / inflammasome | |||||||||
Function / homology | Function and homology information bacterial-type flagellum filament / IPAF inflammasome complex / entry of bacterium into host cell / caspase binding / positive regulation of protein processing / protein serine/threonine kinase binding / pyroptosis / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / bacterial-type flagellum-dependent cell motility / endopeptidase activator activity ...bacterial-type flagellum filament / IPAF inflammasome complex / entry of bacterium into host cell / caspase binding / positive regulation of protein processing / protein serine/threonine kinase binding / pyroptosis / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / bacterial-type flagellum-dependent cell motility / endopeptidase activator activity / negative regulation of tumor necrosis factor-mediated signaling pathway / activation of innate immune response / detection of bacterium / positive regulation of interleukin-1 beta production / positive regulation of JNK cascade / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process / perikaryon / regulation of apoptotic process / defense response to Gram-negative bacterium / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / innate immune response / intracellular membrane-bounded organelle / neuronal cell body / apoptotic process / structural molecule activity / negative regulation of apoptotic process / protein homodimerization activity / extracellular region / ATP binding / identical protein binding / metal ion binding / plasma membrane / cytosol Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) / Salmonella enterica subsp. enterica serovar Typhimurium (bacteria) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 40.0 Å | |||||||||
Authors | Diebolder CA / Halff EF / Koster AJ / Huizinga EG / Koning RI | |||||||||
Citation | Journal: Structure / Year: 2015 Title: Cryoelectron Tomography of the NAIP5/NLRC4 Inflammasome: Implications for NLR Activation. Authors: Christoph A Diebolder / Els F Halff / Abraham J Koster / Eric G Huizinga / Roman I Koning / Abstract: Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from ...Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here, we show by cryoelectron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a pitch of 6.5 nm. Subtomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight into the conformational changes that accompany NLR activation. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2901.map.gz | 562.1 KB | EMDB map data format | |
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Header (meta data) | emd-2901-v30.xml emd-2901.xml | 13 KB 13 KB | Display Display | EMDB header |
Images | EMD-2901.png emd_2901.png | 1.3 MB 1.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2901 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2901 | HTTPS FTP |
-Related structure data
Related structure data | 5aj2MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2901.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | sub tomogram average of NAIP5/NLRC4 polymer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 5.463 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : NAIP5/NLRC4/FliC-D0L multimer
Entire | Name: NAIP5/NLRC4/FliC-D0L multimer |
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Components |
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-Supramolecule #1000: NAIP5/NLRC4/FliC-D0L multimer
Supramolecule | Name: NAIP5/NLRC4/FliC-D0L multimer / type: sample / ID: 1000 / Details: NLRC4 is the main compound within the complex / Oligomeric state: multimer / Number unique components: 3 |
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-Macromolecule #1: NLR family CARD domain-containing protein 4
Macromolecule | Name: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 1 / Name.synonym: NLRC4 / Oligomeric state: multimer / Recombinant expression: Yes |
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Source (natural) | Organism: Mus musculus (house mouse) / synonym: mouse |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE |
Sequence | UniProtKB: NLR family CARD domain-containing protein 4 |
-Macromolecule #2: Baculoviral IAP repeat-containing protein 5
Macromolecule | Name: Baculoviral IAP repeat-containing protein 5 / type: protein_or_peptide / ID: 2 / Name.synonym: NAIP5 / Oligomeric state: multimer / Recombinant expression: Yes |
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Source (natural) | Organism: Mus musculus (house mouse) / synonym: mouse |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE |
Sequence | UniProtKB: Baculoviral IAP repeat-containing protein 1e |
-Macromolecule #3: Flagellin
Macromolecule | Name: Flagellin / type: protein_or_peptide / ID: 3 / Name.synonym: FliC-D0L / Oligomeric state: multimer / Recombinant expression: Yes |
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Source (natural) | Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE |
Sequence | UniProtKB: Flagellin |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | helical array |
-Sample preparation
Concentration | 1.2 mg/mL |
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Buffer | pH: 7.5 / Details: 100 mM NaCl, 20 mM HEPES, 2mM Benzamidin, 2mM DTT |
Grid | Details: glow discharged Cu 200 mesh quantifoil |
Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 95 % / Instrument: LEICA EM GP / Method: 3 seconds blotting |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.5 µm / Nominal defocus min: 6.5 µm / Nominal magnification: 18000 |
Specialist optics | Energy filter - Name: GATAN quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 50.0 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -66 ° / Tilt series - Axis1 - Max angle: 66 ° |
Date | May 21, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 67 / Average electron dose: 100 e/Å2 / Details: 16 single axis tilt series / Bits/pixel: 16 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: TOMOCTF |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 5.57 Å Applied symmetry - Helical parameters - Δ&Phi: 30.9 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: IMOD, PEET / Number subtomograms used: 50 |
Details | filaments were traced by hand along the outer rim, resulting in an initial protomer model. |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: SITUS |
Details | SITUS collage was used for simultaneous multi fragment refinement of symmetry related NLRC4 monomers. Each NLR constisted of 3 rigid bodies: LRR, NBD-HDI and WHD-HD2. CARD was excluded because it did not follow identical helical symmetry |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-5aj2: |