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
Deposition
Feb 17, 2015
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Header (metadata) release
Mar 11, 2015
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Map release
Nov 11, 2015
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Update
Jun 29, 2016
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Current status
Jun 29, 2016
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
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
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
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GATAN quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 50.0 eV
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
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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: Cryo electron tomography of the Naip5-Nlrc4 inflammasome
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