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- PDB-6b5b: Cryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome -

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Basic information

Entry
Database: PDB / ID: 6b5b
TitleCryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome
Components
  • Baculoviral IAP repeat-containing protein 1e
  • Flagellin
  • NLR family CARD domain-containing protein 4
KeywordsIMMUNE SYSTEM / Innate immunity / molecular complex
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 / detection of bacterium / activation of innate immune response / 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 / NLR family CARD domain-containing protein 4 / Flagellin hook, IN motif / NLRC4, helical domain / Flagellin hook IN motif / 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 / NLR family CARD domain-containing protein 4 / Flagellin hook, IN motif / NLRC4, helical domain / Flagellin hook IN motif / 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)
Legionella pneumophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsTenthorey, J.L. / Haloupek, N. / Lopez-Blanco, J.R. / Grob, P. / Adamson, E. / Hartenian, E. / Lind, N.A. / Bourgeois, N.M. / Chacon, P. / Nogales, E. / Vance, R.E.
Funding support Spain, United States, 3items
OrganizationGrant numberCountry
MinecoBFU2016-76220-P Spain
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI075039 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI063302 United States
CitationJournal: Science / Year: 2017
Title: The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.
Authors: Jeannette L Tenthorey / Nicole Haloupek / José Ramón López-Blanco / Patricia Grob / Elise Adamson / Ella Hartenian / Nicholas A Lind / Natasha M Bourgeois / Pablo Chacón / Eva Nogales / Russell E Vance /
Abstract: Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in ...Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.
History
DepositionSep 29, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 6, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Baculoviral IAP repeat-containing protein 1e
B: NLR family CARD domain-containing protein 4
C: NLR family CARD domain-containing protein 4
F: Flagellin


Theoretical massNumber of molelcules
Total (without water)452,1414
Polymers452,1414
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9000 Å2
ΔGint-41 kcal/mol
Surface area144590 Å2

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Components

#1: Protein Baculoviral IAP repeat-containing protein 1e / Neuronal apoptosis inhibitory protein 5


Mass: 159874.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Naip5 / Production host: Homo sapiens (human) / References: UniProt: Q9R016
#2: Protein NLR family CARD domain-containing protein 4 / Caspase recruitment domain-containing protein 12 / Ice protease-activating factor / Ipaf


Mass: 116886.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Nlrc4 / Production host: Homo sapiens (human) / References: UniProt: Q3UP24
#3: Protein Flagellin /


Mass: 58494.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila (bacteria) / Gene: fliC / Production host: Homo sapiens (human) / References: UniProt: G8UUW9, UniProt: Q5ZVV0*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1NAIP5-NLRC4-flagellin inflammasomeCOMPLEXall0MULTIPLE SOURCES
2NAIP5COMPLEX#11RECOMBINANT
3NLRC4COMPLEX#21RECOMBINANT
4flagellinCOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Mus musculus (house mouse)10090
34Legionella pneumophila (bacteria)446
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
310 mMpotassium chlorideKCl1
45 mMmagnesium chlorideMgCl21
55 %glycerolC3H8O31
60.02 %NP-40(C2H4O)multC1H24O1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Carbon-coated / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1800 nm
Image recordingAverage exposure time: 6 sec. / Electron dose: 45.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
2SerialEMimage acquisition
4Gctf0.5CTF correction
7UCSF Chimera1.10.1model fittingVisualization and rigid body fitting
8ADP_EM1.21model fittingSpherical harmonics-based rigid body fitting
9iMODFIT1.45model fittingNMA-based flexible fitting
11EMAN22.05initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
15PHENIX1.11.1-2575model refinementFinal refinement (phenix.real_space_refine)
16I-TASSER5.1model refinementInitial homology modeling
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 856358
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252214 / Symmetry type: POINT
Atomic model buildingB value: 167 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Details: Homology models predicted by I-TASSER server were used as initial model sources. The main structural template identified by I-TASSER for NAIP5 was the crystal structure of NLRC4 in the ...Details: Homology models predicted by I-TASSER server were used as initial model sources. The main structural template identified by I-TASSER for NAIP5 was the crystal structure of NLRC4 in the inactive conformation (PDB ID: 4KXF) that covered all the domains except the N-terminal BIR region, where homology models from several BIR domains were recognized (PDB IDs: 1SE0, 2VM5, and 1OXQ for BIR1, BIR2, and BIR3, respectively). Predictions were first rigid body docked into the density map using Chimera and/or ADP_EM. Then, the docked models were flexibly fitted with iMODFIT, if necessary. Finally, all models were refined in Phenix.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00425039
ELECTRON MICROSCOPYf_angle_d1.01333814
ELECTRON MICROSCOPYf_dihedral_angle_d10.15715171
ELECTRON MICROSCOPYf_chiral_restr0.0513839
ELECTRON MICROSCOPYf_plane_restr0.0064313

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