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- EMDB-3142: Electron cryo-microscopy of PrgJ/NAIP2/CARD-truncated NLRC4 infla... -

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Basic information

Entry
Database: EMDB / ID: EMD-3142
TitleElectron cryo-microscopy of PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome with pseudo c10 symmetry
Map dataReconstruction of PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome with pseudo c10 symmetry
Sample
  • Sample: PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome
  • Protein or peptide: NLRC4
  • Protein or peptide: PrgJ
  • Protein or peptide: NAIP2
Keywordsinflammasome / NLRC4 / NAIP
Biological speciesMus musculus (house mouse) / Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 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) releaseOct 28, 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: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3142.png

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Map

FileDownload / File: emd_3142.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome with pseudo c10 symmetry
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.01337608 - 0.03338128
Average (Standard dev.)-0.00000303 (±0.00198181)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0130.033-0.000

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Supplemental data

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Sample components

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Entire : PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome

EntireName: PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome
Components
  • Sample: PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome
  • Protein or peptide: NLRC4
  • Protein or peptide: PrgJ
  • Protein or peptide: NAIP2

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Supramolecule #1000: PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome

SupramoleculeName: PrgJ/NAIP2/CARD-truncated NLRC4 inflammasome / type: sample / ID: 1000
Oligomeric state: 10 copies of heterotrimer of PrgJ, NAIP2 and CARD-truncated NLRC4
Number unique components: 3
Molecular weightExperimental: 1.0 MDa / Theoretical: 1.0 MDa

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Macromolecule #1: NLRC4

MacromoleculeName: NLRC4 / type: protein_or_peptide / ID: 1 / Name.synonym: IPAF / Number of copies: 10 / 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: PrgJ

MacromoleculeName: PrgJ / 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: NAIP2

MacromoleculeName: NAIP2 / 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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8 / Details: 10 mM Tris-HCl pH 8.0, 100 mM NaCl and 10 mM DTT
GridDetails: 200 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 110 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 second

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37879 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 22,500 times magnification
DateMay 26, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2039 / Average electron dose: 46 e/Å2
Details: Every image is the average of 32 frames recorded by the direct electron detector
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C10 (10 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION1.3 / Number images used: 3233
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4kxf

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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