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- EMDB-3143: Electron microscopy of flagellin/NAIP5/CARD-truncated NLRC4(R288A) -

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Basic information

Entry
Database: EMDB / ID: EMD-3143
TitleElectron microscopy of flagellin/NAIP5/CARD-truncated NLRC4(R288A)
Map dataReconstruction of flagellin/NAIP5/CARD-truncated NLRC4(R288A)
Sample
  • Sample: Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome
  • Protein or peptide: NLRC4
  • Protein or peptide: Flagellin
  • Protein or peptide: NAIP5
Keywordsinflammasome / NLRC4 / NAIP
Biological speciesMus musculus (house mouse) / Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 25.2 Å
AuthorsHu ZH / Zhou Q / Zhang CL / Fan SL / Cheng W / Zhao Y / Shao F / Wang HW / Sui SF / Chai JJ
CitationJournal: Science / Year: 2015
Title: Structural and biochemical basis for induced self-propagation of NLRC4.
Authors: Zehan Hu / Qiang Zhou / Chenlu Zhang / Shilong Fan / Wei Cheng / Yue Zhao / Feng Shao / Hong-Wei Wang / Sen-Fang Sui / Jijie Chai /
Abstract: Responding to stimuli, nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) oligomerize into multiprotein complexes, termed inflammasomes, mediating innate immunity. ...Responding to stimuli, nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) oligomerize into multiprotein complexes, termed inflammasomes, mediating innate immunity. Recognition of bacterial pathogens by NLR apoptosis inhibitory proteins (NAIPs) induces NLR family CARD domain-containing protein 4 (NLRC4) activation and formation of NAIP-NLRC4 inflammasomes. The wheel-like structure of a PrgJ-NAIP2-NLRC4 complex determined by cryogenic electron microscopy at 6.6 angstrom reveals that NLRC4 activation involves substantial structural reorganization that creates one oligomerization surface (catalytic surface). Once activated, NLRC4 uses this surface to catalyze the activation of an inactive NLRC4, self-propagating its active conformation to form the wheel-like architecture. NAIP proteins possess a catalytic surface matching the other oligomerization surface (receptor surface) of NLRC4 but not those of their own, ensuring that one NAIP is sufficient to initiate NLRC4 oligomerization.
History
DepositionSep 4, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseOct 28, 2015-
UpdateOct 28, 2015-
Current statusOct 28, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3143.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of flagellin/NAIP5/CARD-truncated NLRC4(R288A)
Voxel sizeX=Y=Z: 4.2 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.12
Minimum - Maximum-0.17053986 - 0.43571782
Average (Standard dev.)-0.00008434 (±0.04080318)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.1710.436-0.000

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Supplemental data

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Sample components

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Entire : Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome

EntireName: Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome
Components
  • Sample: Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome
  • Protein or peptide: NLRC4
  • Protein or peptide: Flagellin
  • Protein or peptide: NAIP5

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Supramolecule #1000: Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome

SupramoleculeName: Flagellin/NAIP5/CARD-truncated NLRC4(R288A) inflammasome
type: sample / ID: 1000 / Oligomeric state: Flagellin:NAIP5:NLRC4 = 1:1:1 / Number unique components: 3
Molecular weightExperimental: 250 KDa / Theoretical: 250 KDa

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Macromolecule #1: NLRC4

MacromoleculeName: NLRC4 / type: protein_or_peptide / ID: 1 / Name.synonym: IPAF / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf21 / Recombinant plasmid: pFastBac 1

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Macromolecule #2: Flagellin

MacromoleculeName: Flagellin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf21 / Recombinant plasmid: pFastBac 1

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Macromolecule #3: NAIP5

MacromoleculeName: NAIP5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf21 / Recombinant plasmid: pFastBac 1

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8 / Details: 25 mM Tris-HCl pH 8.0 and 150 mM NaCl
StainingType: NEGATIVE
Details: Grids with adsorbed protein staind with 2% w/v uranyl acetate for 60 seconds.
GridDetails: 200 mesh copper grid with thin carbon film
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 133588 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateDec 22, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 106
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.2 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION1.3 / Number images used: 2734
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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