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- PDB-6mks: Cryo-EM structure of NLRC4-CARD filament -

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Basic information

Entry
Database: PDB / ID: 6mks
TitleCryo-EM structure of NLRC4-CARD filament
ComponentsChimera protein of NLR family CARD domain-containing protein 4 and EGFP
KeywordsPROTEIN FIBRIL / NLRC4 / Helical assembly / Inflammasome
Function / homology
Function and homology information


pyroptosis / IPAF inflammasome complex / caspase binding / positive regulation of protein processing / activation of cysteine-type endopeptidase activity / interleukin-1 beta production / activation of innate immune response / positive regulation of interleukin-1 beta production / endopeptidase activator activity / bioluminescence ...pyroptosis / IPAF inflammasome complex / caspase binding / positive regulation of protein processing / activation of cysteine-type endopeptidase activity / interleukin-1 beta production / activation of innate immune response / positive regulation of interleukin-1 beta production / endopeptidase activator activity / bioluminescence / detection of bacterium / generation of precursor metabolites and energy / protein homooligomerization / activation of cysteine-type endopeptidase activity involved in apoptotic process / regulation of apoptotic process / positive regulation of NF-kappaB transcription factor activity / inflammatory response / defense response to bacterium / positive regulation of apoptotic process / apoptotic process / innate immune response / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / cytosol
Caspase recruitment domain / Green fluorescent protein-related / Green fluorescent protein / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Green fluorescent protein, GFP / CARD domain / NACHT nucleoside triphosphatase ...Caspase recruitment domain / Green fluorescent protein-related / Green fluorescent protein / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Green fluorescent protein, GFP / CARD domain / NACHT nucleoside triphosphatase / Green fluorescent protein / Death-like domain superfamily
EGFP / NLR family CARD domain-containing protein 4
Biological speciesHomo sapiens (human)
Vaccinia virus
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZheng, W. / Matyszewski, M. / Sohn, J. / Egelman, E.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM0352569 United States
American Cancer SocietyRG-15-224 United States
CitationJournal: J Biol Chem / Year: 2018
Title: Cryo-EM structure of the NLRC4 filament provides insights into how symmetric and asymmetric supramolecular structures drive inflammasome assembly.
Authors: Mariusz Matyszewski / Weili Zheng / Jacob Lueck / Brendan Antiochos / Edward H Egelman / Jungsan Sohn /
Abstract: Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The NOD-like receptor (NLR) family apoptosis inhibitory protein (NAIP)·NLR family ...Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The NOD-like receptor (NLR) family apoptosis inhibitory protein (NAIP)·NLR family caspase-recruiting domain (CARD) domain-containing 4 (NLRC4) inflammasome recognizes intracellular bacteria and induces the polymerization of the caspase-1 protease, which in turn executes maturation of interleukin-1β (IL-1β) and pyroptosis. Several high-resolution structures of the fully assembled NAIP·NLRC4 complex are available, but these structures do not resolve the architecture of the CARD filament in atomic detail. Here, we present the cryo-EM structure of the filament assembled by the CARD of human NLRC4 (NLRC4) at 3.4 Å resolution. The structure revealed that the helical architecture of the NLRC4 filament is essentially identical to that of the downstream filament assembled by the CARD of caspase-1 (casp1), but deviates from the split washer-like assembly of the NAIP·NLRC4 oligomer. Our results suggest that architectural complementarity is a major driver for the recognition between upstream and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4 filament assembly rationalized why an (un)decameric NLRC4 oligomer is optimal for assembling the helical base of the NLRC4 filament. Together, our results explain how symmetric and asymmetric supramolecular assemblies enable high-fidelity signaling in inflammasomes.
Validation Report
SummaryFull reportAbout validation report
History
DepositionSep 26, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 7, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 14, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_DOI ..._citation.journal_abbrev / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 9, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
A: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
B: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
C: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
D: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
E: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
F: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
G: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
H: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
I: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
J: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
K: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
L: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
M: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
N: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
O: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
P: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
Q: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
R: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
S: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
T: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
U: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
V: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
W: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
X: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
Y: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
Z: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
a: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
b: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
c: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
d: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
e: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP


Theoretical massNumber of molelcules
Total (without water)1,198,73331
Polymers1,198,73331
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Chimera protein of NLR family CARD domain-containing protein 4 and EGFP / CARD / LRR / and NACHT-containing protein / Clan protein / Caspase recruitment domain-containing ...CARD / LRR / and NACHT-containing protein / Clan protein / Caspase recruitment domain-containing protein 12 / Ice protease-activating factor / Ipaf


Mass: 38668.805 Da / Num. of mol.: 31
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Vaccinia virus
Gene: NLRC4, CARD12, CLAN, CLAN1, IPAF, UNQ6189/PRO20215, EGFP
Plasmid: pET21b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9NPP4, UniProt: A0A1V0D974

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: NLRC4-CARD filament / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET21b
Buffer solutionpH: 7
Details: 20mM HEPES at pH 7.4, 400mM NaCl, 10% glycerol, 1mM EDTA and 1mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1690

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Processing

SoftwareName: PHENIX / Version: dev_2919: / Classification: refinement
EM software
IDNameCategory
1EMAN2particle selection
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
8RosettaEMmodel fitting
13SPIDER3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 100.6 ° / Axial rise/subunit: 5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 402078
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 299537 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 4IKM

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