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- EMDB-2901: Cryo electron tomography of Naip5/Nlrc4 inflammasome -

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Basic information

Entry
Database: EMDB / ID: EMD-2901
TitleCryo electron tomography of Naip5/Nlrc4 inflammasome
Map datasub tomogram average of NAIP5/NLRC4 polymer
Sample
  • Sample: NAIP5/NLRC4/FliC-D0L multimer
  • Protein or peptide: NLR family CARD domain-containing protein 4
  • Protein or peptide: Baculoviral IAP repeat-containing protein 5
  • Protein or peptide: Flagellin
KeywordsNLRs / 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
Baculoviral IAP repeat-containing protein 1 / Flagellin D3 / Flagellin D3 domain / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region ...Baculoviral IAP repeat-containing protein 1 / Flagellin D3 / Flagellin D3 domain / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region / NOD2, winged helix domain / NOD2 winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / BIR repeat. / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NLR family CARD domain-containing protein 4 / Flagellin / Baculoviral IAP repeat-containing protein 1e
Similarity search - Component
Biological speciesMus musculus (house mouse) / Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsDiebolder CA / Halff EF / Koster AJ / Huizinga EG / Koning RI
CitationJournal: 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
DepositionFeb 17, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseNov 11, 2015-
UpdateJun 29, 2016-
Current statusJun 29, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 175
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 175
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5aj2
  • Surface level: 175
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5aj2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2901.png

emd_2901.png

Downloads & links

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Map

FileDownload / File: emd_2901.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsub tomogram average of NAIP5/NLRC4 polymer
Voxel sizeX=Y=Z: 5.463 Å
Density
Contour LevelBy AUTHOR: 175.0 / Movie #1: 175
Minimum - Maximum0.0 - 255.0
Average (Standard dev.)16.017105099999998 (±46.346935270000003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-410
Dimensions100100100
Spacing100100100
CellA=B=C: 546.3 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.4635.4635.463
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z546.300546.300546.300
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-4010
NC/NR/NS100100100
D min/max/mean0.000255.00016.017

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Supplemental data

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Sample components

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Entire : NAIP5/NLRC4/FliC-D0L multimer

EntireName: NAIP5/NLRC4/FliC-D0L multimer
Components
  • Sample: NAIP5/NLRC4/FliC-D0L multimer
  • Protein or peptide: NLR family CARD domain-containing protein 4
  • Protein or peptide: Baculoviral IAP repeat-containing protein 5
  • Protein or peptide: Flagellin

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Supramolecule #1000: NAIP5/NLRC4/FliC-D0L multimer

SupramoleculeName: 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

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 1 / Name.synonym: NLRC4 / Oligomeric state: multimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE
SequenceUniProtKB: NLR family CARD domain-containing protein 4

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Macromolecule #2: Baculoviral IAP repeat-containing protein 5

MacromoleculeName: Baculoviral IAP repeat-containing protein 5 / type: protein_or_peptide / ID: 2 / Name.synonym: NAIP5 / Oligomeric state: multimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE
SequenceUniProtKB: Baculoviral IAP repeat-containing protein 1e

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Macromolecule #3: Flagellin

MacromoleculeName: Flagellin / type: protein_or_peptide / ID: 3 / Name.synonym: FliC-D0L / Oligomeric state: multimer / Recombinant expression: Yes
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293E / Recombinant plasmid: pUPE
SequenceUniProtKB: Flagellin

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statehelical array

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5 / Details: 100 mM NaCl, 20 mM HEPES, 2mM Benzamidin, 2mM DTT
GridDetails: glow discharged Cu 200 mesh quantifoil
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 95 % / Instrument: LEICA EM GP / Method: 3 seconds blotting

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 opticsEnergy filter - Name: GATAN quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 50.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -66 ° / Tilt series - Axis1 - Max angle: 66 °
DateMay 21, 2013
Image recordingCategory: 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

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Image processing

CTF correctionDetails: TOMOCTF
Final reconstructionApplied 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
Detailsfilaments were traced by hand along the outer rim, resulting in an initial protomer model.

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Atomic model buiding 1

Initial modelPDB ID:

4kxf

SoftwareName: SITUS
DetailsSITUS 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
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5aj2:
Cryo electron tomography of the Naip5-Nlrc4 inflammasome

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