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- PDB-8fml: Cryo-EM structure of NLR family apoptosis inhibitory protein 5 (N... -

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Basic information

Entry
Database: PDB / ID: 8fml
TitleCryo-EM structure of NLR family apoptosis inhibitory protein 5 (NAIP5) in complex with a full-length flagellin (FliC) ligand
Components
  • Baculoviral IAP repeat-containing protein 1e
  • Flagellin
KeywordsIMMUNE SYSTEM / Inflammasome / Innate immunity / Bacterial ligand / host-pathogen interaction / Protein complex
Function / homology
Function and homology information


TLR5 cascade / MyD88 cascade initiated on plasma membrane / NFkB and MAPK activation mediated by TRAF6 / IPAF inflammasome complex / The IPAF inflammasome / bacterial-type flagellum / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / positive regulation of interleukin-1 beta production ...TLR5 cascade / MyD88 cascade initiated on plasma membrane / NFkB and MAPK activation mediated by TRAF6 / IPAF inflammasome complex / The IPAF inflammasome / bacterial-type flagellum / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / detection of bacterium / positive regulation of interleukin-1 beta production / perikaryon / defense response to Gram-negative bacterium / receptor ligand activity / defense response to bacterium / inflammatory response / symbiont entry into host cell / innate immune response / neuronal cell body / negative regulation of apoptotic process / apoptotic process / structural molecule activity / extracellular space / ATP binding / metal ion binding
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / Flagellin D3 / : / Flagellin D3 domain / Flagellin, barrel domain / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Flagellin, C-terminal domain, subdomain 2 ...Baculoviral IAP repeat-containing protein 1 / Flagellin D3 / : / Flagellin D3 domain / Flagellin, barrel domain / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Flagellin, C-terminal domain, subdomain 2 / Flagellin, C-terminal domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region / 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 / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Flagellin / Baculoviral IAP repeat-containing protein 1e
Similarity search - Component
Biological speciesMus musculus (house mouse)
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsPaidimuddala, B. / Zhang, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis for flagellin-induced NAIP5 activation.
Authors: Bhaskar Paidimuddala / Jianhao Cao / Liman Zhang /
Abstract: The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain ...The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain of NAIP forms a steric clash with NLRC4 to open it up. However, how ligand binding activates NAIP is less clear. Here, we investigated the dynamics of the ligand-binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.9-Å resolution. The structure revealed a "trap and lock" mechanism in FliC recognition, whereby FliC-D0 is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by ID (insertion domain) and C-terminal tail of NAIP5. The FliC-D0 domain further inserts into ID to stabilize the complex. According to this mechanism, FliC triggers the conformational change of NAIP5 by bringing multiple flexible domains together.
History
DepositionDec 23, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Baculoviral IAP repeat-containing protein 1e
B: Flagellin


Theoretical massNumber of molelcules
Total (without water)213,7432
Polymers213,7432
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 161128.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Naip5, Birc1e, Naip-rs3 / Plasmid: pcDNA3.4-flag-NAIP5 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9R016
#2: Protein Flagellin / Phase 1-I flagellin


Mass: 52614.418 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Gene: fliC, flaF, hag, STM1959 / Plasmid: pCMV-His-fliC / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P06179

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: NAIP5 in complex with a full-length bacterial flagellin ligand (FliC)
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.2135 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4616
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
UCSF ChimeraX1.1/v9model building
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Coot0.9.4.1 ELmodel fitting
9PHENIX1.20.1-4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199022 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411827
ELECTRON MICROSCOPYf_angle_d0.76515993
ELECTRON MICROSCOPYf_dihedral_angle_d5.821554
ELECTRON MICROSCOPYf_chiral_restr0.0461791
ELECTRON MICROSCOPYf_plane_restr0.0062061

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