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- EMDB-6460: Cryo-EM Structure of the Activated NAIP2/NLRC4 Inflammasome Revea... -

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Basic information

Entry
Database: EMDB / ID: EMD-6460
TitleCryo-EM Structure of the Activated NAIP2/NLRC4 Inflammasome Reveals Nucleated Polymerization
Map dataReconstruction of 10-fold NAIP2/NLRC4 inflammasome
Sample
  • Sample: NAIP2/NLRC4 inflammasome, 10-fold disk
  • Protein or peptide: NLR family CARD domain-containing protein 4
  • Protein or peptide: NLR family, apoptosis inhibitory protein 2
KeywordsInflammasome / NLRC4 / NAIP2
Function / homology
Function and homology information


IPAF inflammasome complex / pyroptotic inflammatory response / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / regulation of apoptotic process / defense response to bacterium / inflammatory response / innate immune response ...IPAF inflammasome complex / pyroptotic inflammatory response / detection of bacterium / activation of innate immune response / positive regulation of interleukin-1 beta production / protein homooligomerization / regulation of apoptotic process / defense response to bacterium / inflammatory response / innate immune response / apoptotic process / protein homodimerization activity / ATP binding / identical protein binding / cytosol
Similarity search - Function
NLR family CARD domain-containing protein 4 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. ...NLR family CARD domain-containing protein 4 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.5 Å
AuthorsZhang L / Chen S / Ruan J / Wu J / Tong AB / Yin Q / Li Y / David L / Lu A / Wang WL ...Zhang L / Chen S / Ruan J / Wu J / Tong AB / Yin Q / Li Y / David L / Lu A / Wang WL / Marks C / Ouyang Q / Zhang X / Mao Y / Wu H
CitationJournal: Science / Year: 2015
Title: Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization.
Authors: Liman Zhang / Shuobing Chen / Jianbin Ruan / Jiayi Wu / Alexander B Tong / Qian Yin / Yang Li / Liron David / Alvin Lu / Wei Li Wang / Carolyn Marks / Qi Ouyang / Xinzheng Zhang / Youdong Mao / Hao Wu /
Abstract: The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the ...The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo-electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes.
History
DepositionSep 5, 2015-
Header (metadata) releaseOct 14, 2015-
Map releaseOct 21, 2015-
UpdateNov 4, 2015-
Current statusNov 4, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6460.tif

Downloads & links

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Map

FileDownload / File: emd_6460.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 10-fold NAIP2/NLRC4 inflammasome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.72 Å/pix.
x 250 pix.
= 430. Å		1.72 Å/pix.
x 250 pix.
= 430. Å		1.72 Å/pix.
x 250 pix.
= 430. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.01187289 - 0.02465408
Average (Standard dev.)0.00017569 (±0.00186498)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 430.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.721.721.72
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z430.000430.000430.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0120.0250.000

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Supplemental data

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Sample components

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Entire : NAIP2/NLRC4 inflammasome, 10-fold disk

EntireName: NAIP2/NLRC4 inflammasome, 10-fold disk
Components
  • Sample: NAIP2/NLRC4 inflammasome, 10-fold disk
  • Protein or peptide: NLR family CARD domain-containing protein 4
  • Protein or peptide: NLR family, apoptosis inhibitory protein 2

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Supramolecule #1000: NAIP2/NLRC4 inflammasome, 10-fold disk

SupramoleculeName: NAIP2/NLRC4 inflammasome, 10-fold disk / type: sample / ID: 1000
Details: The sample contains multiple oligomeric states, including 10-fold, 11-fold, and 12-fold disks.
Oligomeric state: One NAIP2 subunit and nine NLRC subunits / Number unique components: 2
Molecular weightExperimental: 1.6 MDa / Theoretical: 1.6 MDa

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Macromolecule #1: NLR family CARD domain-containing protein 4

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 1 / Name.synonym: NLRC4
Details: One activated NAIP2 induces polymerization of NLRC4.
Number of copies: 9
Oligomeric state: NLRC4 inflammasome comprises nine NLRC4 and one NAIP2
Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / Strain: RAW 264.7 / synonym: mouse
Tissue: Abelson murine leukemia virus-induced tumor; ascites
Cell: macrophage / Location in cell: cytosol
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: IPLB-SF21-AE / Recombinant cell: Sf21 / Recombinant plasmid: pFastBac1
SequenceUniProtKB: NLR family CARD domain-containing protein 4

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Macromolecule #2: NLR family, apoptosis inhibitory protein 2

MacromoleculeName: NLR family, apoptosis inhibitory protein 2 / type: protein_or_peptide / ID: 2 / Name.synonym: NAIP2
Details: One activated NAIP2 induces polymerization of NLRC4.
Number of copies: 1
Oligomeric state: NLRC4 inflammasome comprises one NAIP2 and nine NLRC4
Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / Strain: RAW 264.7 / synonym: mouse
Tissue: Abelson murine leukemia virus-induced tumor; ascites
Cell: macrophage / Location in cell: cytosol
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: IPLB-SF21-AE / Recombinant cell: Sf21 / Recombinant plasmid: pFastBac1

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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

Concentration3 mg/mL
BufferpH: 7.5 / Details: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM DTT
GridDetails: 400 mesh C-flat grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 103 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 seconds before plunging

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Electron microscopy

MicroscopeOTHER
TemperatureMin: 79.5 K / Max: 80 K / Average: 79.5 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 21,000 times magnification.
DateApr 1, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 9113 / Average electron dose: 48 e/Å2
Details: average of 36 frames recorded by the direct electron detector
Bits/pixel: 32
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 28736 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 21000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

CTF correctionDetails: Wiener-type filter
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.5 Å / Resolution method: OTHER / Software - Name: Spider, EMAN2, Relion / Number images used: 4489

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