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

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Entry
Database: PDB / ID: 5aj2
TitleCryo electron tomography of the Naip5-Nlrc4 inflammasome
Components(NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4) x 3
KeywordsAPOPTOSIS
Function / homology
Function and homology information


IPAF inflammasome complex / caspase binding / positive regulation of protein processing / pyroptosis / endopeptidase activator activity / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process ...IPAF inflammasome complex / caspase binding / positive regulation of protein processing / pyroptosis / endopeptidase activator activity / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process / regulation of apoptotic process / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / innate immune response / intracellular membrane-bounded organelle / apoptotic process / protein homodimerization activity / ATP binding / identical protein binding / plasma membrane / cytosol
Similarity search - Function
NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily ...NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / 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
Similarity search - Component
Biological speciesMUS MUSCULUS (house mouse)
MethodELECTRON MICROSCOPY / electron tomography / cryo EM / Resolution: 40 Å
Model type detailsCA ATOMS ONLY, CHAIN A, C, B
AuthorsDiebolder, C.A. / Halff, E.F. / Koster, A.J. / Huizinga, E.G. / Koning, R.I.
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 20, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 11, 2015Provider: repository / Type: Initial release
Revision 1.1May 18, 2016Group: Database references
Revision 1.2Aug 23, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4
B: NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4
C: NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4


Theoretical massNumber of molelcules
Total (without water)117,0503
Polymers117,0503
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Noncrystallographic symmetry (NCS)NCS oper: (Code: given / Matrix: (0.858065, 0.513541), (1), (-0.513541, 0.858065) / Vector: -131.7016, 5.57, 173.487)

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Components

#1: Protein NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4 / CASPASE RECRUITMENT DOMAIN-CONTAINING PROTEIN 12 / ICE PROTEASE-ACTIVATING FACTOR / IPAF / Coordinate model: Cα atoms only


Mass: 40914.316 Da / Num. of mol.: 1 / Fragment: RESIDUES 1-355
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) MUS MUSCULUS (house mouse) / Plasmid: PUPE / Cell line (production host): HEK293E / Production host: HOMO SAPIENS (human) / References: UniProt: Q3UP24
#2: Protein NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4 / CASPASE RECRUITMENT DOMAIN-CONTAINING PROTEIN 12 / ICE PROTEASE-ACTIVATING FACTOR / IPAF / Coordinate model: Cα atoms only


Mass: 25524.809 Da / Num. of mol.: 1 / Fragment: RESIDUES 356-580
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) MUS MUSCULUS (house mouse) / Plasmid: PUPE / Cell line (production host): HEK293E / Production host: HOMO SAPIENS (human) / References: UniProt: Q3UP24
#3: Protein NLR FAMILY CARD DOMAIN-CONTAINING PROTEIN 4 / CASPASE RECRUITMENT DOMAIN-CONTAINING PROTEIN 12 / ICE PROTEASE-ACTIVATING FACTOR / IPAF / Coordinate model: Cα atoms only


Mass: 50610.996 Da / Num. of mol.: 1 / Fragment: RESIDUES 580-1024
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) MUS MUSCULUS (house mouse) / Plasmid: PUPE / Cell line (production host): HEK293E / Production host: HOMO SAPIENS (human) / References: UniProt: Q3UP24

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: electron tomography

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Sample preparation

ComponentName: NAIP5-NLRC4-FLIC-D0L MULTIMER / Type: COMPLEX
Buffer solutionName: 100 MM NACL, 20 MM HEPES, 2MM BENZAMIDIN, 2MM DTT / pH: 7.5 / Details: 100 MM NACL, 20 MM HEPES, 2MM BENZAMIDIN, 2MM DTT
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE-PROPANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE-PROPANE MIXTURE, HUMIDITY- 95, INSTRUMENT- LEICA EM GP, METHOD- 3 SECONDS BLOTTING,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: May 21, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 18000 X / Nominal defocus max: 7500 nm / Nominal defocus min: 6500 nm
Specimen holderTilt angle max: 66 ° / Tilt angle min: -66 °
Image recordingElectron dose: 100 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 67

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Processing

EM software
IDNameCategory
1Situsmodel fitting
2IMOD3D reconstruction
3PEET3D reconstruction
CTF correctionDetails: TOMOCTF
3D reconstructionMethod: CROSS-COMMON LINES / Resolution: 40 Å / Num. of particles: 50 / Nominal pixel size: 5.463 Å / Actual pixel size: 5.463 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2901. (DEPOSITION ID: 13118).
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 4KXF

4kxf

RefinementHighest resolution: 40 Å
Refinement stepCycle: LAST / Highest resolution: 40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms904 0 0 0 904

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