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- PDB-3jbl: Cryo-EM Structure of the Activated NAIP2/NLRC4 Inflammasome Revea... -

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Entry
Database: PDB / ID: 3jbl
TitleCryo-EM Structure of the Activated NAIP2/NLRC4 Inflammasome Reveals Nucleated Polymerization
ComponentsNLR family CARD domain-containing protein 4
KeywordsIMMUNE SYSTEM / Inflammasome / NLRC4 / NAIP2
Function / homology
Function and homology information


IPAF inflammasome complex / caspase binding / positive regulation of protein processing / pyroptosis / endopeptidase activator activity / activation of innate immune response / detection of bacterium / positive regulation of interleukin-1 beta production / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process ...IPAF inflammasome complex / caspase binding / positive regulation of protein processing / pyroptosis / endopeptidase activator activity / activation of innate immune response / detection of bacterium / positive regulation of interleukin-1 beta production / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process / regulation of apoptotic process / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / intracellular membrane-bounded organelle / innate immune response / apoptotic process / protein homodimerization activity / ATP binding / identical protein binding / plasma membrane / cytosol
Similarity search - Function
NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily ...NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsZhang, L. / Chen, S. / Ruan, J. / Wu, J. / Tong, A.B. / Yin, Q. / Li, Y. / David, L. / Lu, A. / Wang, W.L. ...Zhang, L. / Chen, S. / Ruan, J. / Wu, J. / Tong, A.B. / Yin, Q. / Li, Y. / David, L. / Lu, A. / Wang, W.L. / 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, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 21, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 4, 2015Group: Database references / Other
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
K: NLR family CARD domain-containing protein 4
A: NLR family CARD domain-containing protein 4
B: NLR family CARD domain-containing protein 4
C: NLR family CARD domain-containing protein 4
D: NLR family CARD domain-containing protein 4
E: NLR family CARD domain-containing protein 4
F: NLR family CARD domain-containing protein 4
G: NLR family CARD domain-containing protein 4
H: NLR family CARD domain-containing protein 4
I: NLR family CARD domain-containing protein 4
J: NLR family CARD domain-containing protein 4


Theoretical massNumber of molelcules
Total (without water)1,168,63711
Polymers1,168,63711
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA93 - 1024
211chain BB93 - 1024
311chain CC93 - 1024

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Components

#1: Protein
NLR family CARD domain-containing protein 4 / NLRC4 / Caspase recruitment domain-containing protein 12 / Ice protease-activating factor / Ipaf


Mass: 106239.711 Da / Num. of mol.: 11 / Fragment: UNP residues 93-1024, SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: RAW 264.7
Tissue: Abelson murine leukemia virus-induced tumor; Ascites
Cell line: macrophage / Cellular location: cytosol / Gene: Nlrc4, Card12, Ipaf / Plasmid: pFastBac1 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-SF21-AE / References: UniProt: Q3UP24
Sequence detailsCOMPLEX COMPRISES TEN NLRC4 SUBUNITS AND ONE NAIP2 SUBUNIT, BUT ELEVEN NLRC4 SUBUNITS HAVE BEEN ...COMPLEX COMPRISES TEN NLRC4 SUBUNITS AND ONE NAIP2 SUBUNIT, BUT ELEVEN NLRC4 SUBUNITS HAVE BEEN MODELED INSTEAD.

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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
IDNameTypeDetailsParent-IDSynonym
1NAIP2/NLRC4 inflammasomeCOMPLEXOne NAIP2 subunit and ten NLRC subunits0
2NLR family CARD domain-containing protein 41NLRC4
3NLR family, apoptosis inhibitory protein 21NAIP2
Molecular weightValue: 1.8 MDa / Experimental value: NO
Buffer solutionName: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM DTT / pH: 8 / Details: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM DTT
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh C-flat grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 103 K / Humidity: 100 %
Details: Blotted for one second before plunging into liquid ethane (FEI VITROBOT MARK IV).
Method: Blot for one second before plunging

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA / Date: Apr 1, 2015 / Details: Tecnai Arctica
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 21000 X / Calibrated magnification: 28736 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 21,000 times magnification.
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 79.5 K / Temperature (max): 80 K / Temperature (min): 79.5 K
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) / Details: 36 frames per exposure, 9 seconds
Image scansNum. digital images: 9113
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
PDB_EXTRACT3.15data extraction
EM software
IDNameVersionCategory
1EMAN23D reconstruction
2RELION3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: Wiener-type filter
SymmetryPoint symmetry: C11 (11 fold cyclic)
3D reconstructionMethod: Maximum likelihood-based projection matching and direct Fourier reconstruction
Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75114 / Nominal pixel size: 0.86 Å / Actual pixel size: 0.86 Å / Details: (Single particle--Applied symmetry: C11) / Symmetry type: POINT
RefinementResolution: 4.5→199.52 Å / SU ML: 1.71 / σ(F): 0 / Phase error: 68.84 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.3828 1400 2.08 %
Rwork0.3643 66009 -
obs0.3647 67409 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 879.99 Å2 / Biso mean: 449.3424 Å2 / Biso min: 42.15 Å2
Refinement stepCycle: LAST / Resolution: 4.5→199.52 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms80124 0 0 0 80124
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00722269
ELECTRON MICROSCOPYf_angle_d2.04830072
ELECTRON MICROSCOPYf_chiral_restr0.0823420
ELECTRON MICROSCOPYf_plane_restr0.0093825
ELECTRON MICROSCOPYf_dihedral_angle_d14.9038316
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A13327ELECTRON MICROSCOPY5.968TORSIONAL
12B13327ELECTRON MICROSCOPY5.968TORSIONAL
13C13327ELECTRON MICROSCOPY5.968TORSIONAL
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
4.5002-4.6610.52951420.506266216763100
4.661-4.84760.5181400.522565796719100
4.8476-5.06830.58291420.522865606702100
5.0683-5.33550.55181440.507967256869100
5.3355-5.66980.5181420.513765686710100
5.6698-6.10760.50641320.52366016733100
6.1076-6.72230.57471400.511466006740100
6.7223-7.6950.55831390.518966026741100
7.695-9.69490.4841330.566486781100
9.6949-199.71630.26731460.22646505665198
Refinement TLS params.

Method: refined / Refine-ID: ELECTRON MICROSCOPY

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.6483-0.0148-0.81610.5416-0.02971.0415-0.0921-0.2760.720.91790.91010.1286-0.4935-0.99386.5976-2.53441.06671.45220.1975-0.1915-0.9965132.5132169.9433227.345
20.33920.0326-0.4781.5798-1.25321.5260.67140.36570.8328-0.11390.53512.1392-0.0754-1.00092.53070.57220.2594-0.1480.263-0.03052.5418118.637170.0307203.5637
30.91810.28850.18080.17870.37332.43170.02980.4883-0.8154-0.82250.4554-0.03060.4049-0.50594.06272.990.7196-0.71070.8932-0.8579-0.7011111.4445163.1167188.73
40.1911-0.16640.13090.146-0.07930.43010.0726-0.06030.0669-0.03440.06990.0441-0.13250.35290.3656.0637-0.03351.00735.14552.34022.82380.3828151.5603199.0583
51.72340.0019-0.31980.09320.09130.1618-0.65970.06950.33830.3025-0.3880.2928-1.4788-0.1943-3.39134.95771.72620.76110.4565-1.66196.013580.3901165.9284193.6584
60.1260.2975-0.40111.0725-2.07595.5533-0.97191.7199-0.6633-2.57560.4051.3923.4934-2.7225-3.69262.85390.85360.27972.4763-1.16226.776373.4334133.8407181.9303
72.0025-0.07791.04252.50320.83212.90821.8728-1.0474-0.84320.37012.05210.84651.2689-1.20818.67190.1423-1.8771-2.37380.8811-0.9823-1.2409149.8426139.3023224.396
81.0995-0.30330.67731.0550.55912.70621.0997-0.106-0.066-0.11220.7275-0.49780.53050.330612.2521-0.59450.35040.0023-0.0104-0.369-1.2496145.0594129.1827193.6455
90.31620.0359-0.05680.3453-0.05610.020.1391-0.23670.0982-0.23770.2298-0.08740.22910.14210.05456.42290.4755-0.31055.95362.54193.1536120.638197.4671199.0473
100.2920.26320.74130.4551.01892.6064-0.6185-0.7641-0.1171-0.19030.10520.0364-3.3111-1.633-0.91333.77021.92390.46292.8651-0.34046.9541112.7285109.4238193.5763
111.2629-0.1782-1.23440.2979-0.18875.1759-0.88860.9514-1.2885-1.2382-1.50020.85833.5394-0.4377-8.92122.1053-0.3894-0.67092.58120.70466.8394123.159178.3694181.2971
120.1930.29510.35040.68990.5250.6-0.44290.03840.4253-0.0385-0.18850.1685-0.325-0.1757-0.50592.7119-2.09670.85362.5989-1.62053.9927145.635986.3881194.985
131.0037-0.2746-1.04011.8342-0.68041.53551.31830.44260.98840.25860.70691.3491-0.5132-1.58315.93860.24891.062-0.4404-1.84040.38881.551129.8424205.9742227.3478
140.8280.7506-0.5110.7334-0.64730.7004-0.38370.30020.1433-0.43750.2161-0.1350.6255-0.24280.89020.4077-0.21420.24490.71770.13392.9386118.2045213.893203.5326
150.62810.31440.31820.36920.47590.96420.41120.3158-0.52410.48850.6748-0.50940.14330.16735.5071.9429-0.7623-0.2398-0.0877-0.51810.6217108.5282211.3397188.9523
160.0044-0.0093-0.01690.020.02940.0505-0.0365-0.00910.00190.27480.0064-0.396-0.0308-0.0344-0.0275.9733-0.42632.17947.09570.72863.640976.0386218.8492199.073
170.3511-0.167-0.12290.1649-0.05220.2599-0.3391-0.08930.24990.4084-0.57350.721-1.05880.2926-3.27673.6369-2.0430.2689-0.1424-1.19125.189783.7723230.8514193.6022
181.32940.0095-1.0110.4238-0.46832.1729-0.74351.4528-0.2709-0.1750.2160.6707-0.3086-3.2165-2.0040.62661.6863-1.74281.5936-0.69726.653860.5575207.632181.9155
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1ELECTRON MICROSCOPY1(chain A and resid 93:302)A93 - 302
2ELECTRON MICROSCOPY2(chain A and resid 303:434)A303 - 434
3ELECTRON MICROSCOPY3(chain A and resid 435:523)A435 - 523
4ELECTRON MICROSCOPY4(chain A and resid 524:533)A524 - 533
5ELECTRON MICROSCOPY5(chain A and resid 534:727)A534 - 727
6ELECTRON MICROSCOPY6(chain A and resid 728:1024)A728 - 1024
7ELECTRON MICROSCOPY7(chain B and resid 93:351)B93 - 351
8ELECTRON MICROSCOPY8(chain B and resid 352:523)B352 - 523
9ELECTRON MICROSCOPY9(chain B and resid 524:533)B524 - 533
10ELECTRON MICROSCOPY10(chain B and resid 534:727)B534 - 727
11ELECTRON MICROSCOPY11(chain B and resid 728:1010)B728 - 1010
12ELECTRON MICROSCOPY12(chain B and resid 1011:1024)B1011 - 1024
13ELECTRON MICROSCOPY13(chain C and resid 93:302)C93 - 302
14ELECTRON MICROSCOPY14(chain C and resid 303:433)C303 - 433
15ELECTRON MICROSCOPY15(chain C and resid 434:523)C434 - 523
16ELECTRON MICROSCOPY16(chain C and resid 524:533)C524 - 533
17ELECTRON MICROSCOPY17(chain C and resid 534:727)C534 - 727
18ELECTRON MICROSCOPY18(chain C and resid 728:1024)C728 - 1024

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