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- EMDB-29493: Cryo-EM structure of human Needle/NAIP/NLRC4 (R288A) -

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Basic information

Entry
Database: EMDB / ID: EMD-29493
TitleCryo-EM structure of human Needle/NAIP/NLRC4 (R288A)
Map data
Sample
  • Complex: the complex of Needle, human NAIP and NLRC4
    • Complex: human NAIP and NLRC4
      • Protein or peptide: Baculoviral IAP repeat-containing protein 1
      • Protein or peptide: NLR family CARD domain-containing protein 4
    • Complex: Burkholderia Needle (chimeric with an anthrax toxin lethal factor tag)
      • Protein or peptide: Lethal factor,Type III secretion system protein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ZINC ION
KeywordsNLRC4 / NAIP / Inflammasome / IMMUNE SYSTEM
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / type III protein secretion system complex / positive regulation of protein processing / caspase binding / protein secretion by the type III secretion system / activation of cysteine-type endopeptidase activity ...anthrax lethal factor endopeptidase / IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / type III protein secretion system complex / positive regulation of protein processing / caspase binding / protein secretion by the type III secretion system / activation of cysteine-type endopeptidase activity / pattern recognition receptor signaling pathway / host cell cytosol / cysteine-type endopeptidase inhibitor activity / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / Uptake and function of anthrax toxins / endopeptidase activator activity / negative regulation of tumor necrosis factor-mediated signaling pathway / detection of bacterium / protein serine/threonine kinase binding / activation of innate immune response / : / positive regulation of interleukin-1 beta production / positive regulation of JNK cascade / protein homooligomerization / metalloendopeptidase activity / positive regulation of inflammatory response / metallopeptidase activity / positive regulation of NF-kappaB transcription factor activity / nervous system development / toxin activity / basolateral plasma membrane / regulation of apoptotic process / negative regulation of neuron apoptotic process / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / intracellular membrane-bounded organelle / innate immune response / negative regulation of apoptotic process / apoptotic process / magnesium ion binding / cell surface / protein homodimerization activity / ATP hydrolysis activity / proteolysis / zinc ion binding / extracellular region / ATP binding / identical protein binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily ...Baculoviral IAP repeat-containing protein 1 / NLR family CARD domain-containing protein 4 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily / Type III secretion needle MxiH, YscF, SsaG, EprI, PscF, EscF / Type III secretion system, needle protein / Anthrax toxin, lethal/endema factor / Anthrax toxin, lethal/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain / Anthrax toxin lethal factor (ATLF)-like domain profile. / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / BIR repeat. / BIR repeat / Inhibitor of Apoptosis domain / BIR repeat profile. / Baculoviral inhibition of apoptosis protein repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Metallopeptidase, catalytic domain superfamily / Death-like domain superfamily / Leucine-rich repeat domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Lethal factor / Baculoviral IAP repeat-containing protein 1 / Type III secretion system protein / NLR family CARD domain-containing protein 4
Similarity search - Component
Biological speciesHomo sapiens (human) / Bacillus anthracis (anthrax bacterium) / Burkholderia (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMatico RE / Yu X / Miller R / Somani S / Ricketts MD / Kumar N / Steele RA / Medley Q / Berger S / Faustin B / Sharma S
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.
Authors: Rosalie E Matico / Xiaodi Yu / Robyn Miller / Sandeep Somani / M Daniel Ricketts / Nikit Kumar / Ruth A Steele / Quintus Medley / Scott Berger / Benjamin Faustin / Sujata Sharma /
Abstract: The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in ...The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.
History
DepositionJan 19, 2023-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29493.png

Downloads & links

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Map

FileDownload / File: emd_29493.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 420 pix.
= 398.16 Å		0.95 Å/pix.
x 420 pix.
= 398.16 Å		0.95 Å/pix.
x 420 pix.
= 398.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.948 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.6194728 - 1.0147607
Average (Standard dev.)0.0004821804 (±0.017633457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 398.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29493_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29493_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : the complex of Needle, human NAIP and NLRC4

EntireName: the complex of Needle, human NAIP and NLRC4
Components
  • Complex: the complex of Needle, human NAIP and NLRC4
    • Complex: human NAIP and NLRC4
      • Protein or peptide: Baculoviral IAP repeat-containing protein 1
      • Protein or peptide: NLR family CARD domain-containing protein 4
    • Complex: Burkholderia Needle (chimeric with an anthrax toxin lethal factor tag)
      • Protein or peptide: Lethal factor,Type III secretion system protein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: the complex of Needle, human NAIP and NLRC4

SupramoleculeName: the complex of Needle, human NAIP and NLRC4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: human NAIP and NLRC4

SupramoleculeName: human NAIP and NLRC4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Burkholderia Needle (chimeric with an anthrax toxin lethal factor tag)

SupramoleculeName: Burkholderia Needle (chimeric with an anthrax toxin lethal factor tag)
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)

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Macromolecule #1: Baculoviral IAP repeat-containing protein 1

MacromoleculeName: Baculoviral IAP repeat-containing protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 160.484359 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AEYCFNMATQ QKASDERISQ FDHNLLPELS ALLGLDAVQL AKELEEEEQK ERAKMQKGYN SQMRSEAKRL KTFVTYEPYS SWIPQEMAA AGFYFTGVKS GIQCFCCSLI LFGAGLTRLP IEDHKRFHPD CGFLLNKDVG NIAKYDIRVK NLKSRLRGGK M RYQEEEAR ...String:
AEYCFNMATQ QKASDERISQ FDHNLLPELS ALLGLDAVQL AKELEEEEQK ERAKMQKGYN SQMRSEAKRL KTFVTYEPYS SWIPQEMAA AGFYFTGVKS GIQCFCCSLI LFGAGLTRLP IEDHKRFHPD CGFLLNKDVG NIAKYDIRVK NLKSRLRGGK M RYQEEEAR LASFRNWPFY VQGISPCVLS EAGFVFTGKQ DTVQCFSCGG CLGNWEEGDD PWKEHAKWFP KCEFLRSKKS SE EITQYIQ SYKGFVDITG EHFVNSWVQR ELPMASAYCN DSIFAYEELR LDSFKDWPRE SAVGVAALAK AGLFYTGIKD IVQ CFSCGG CLEKWQEGDD PLDDHTRCFP NCPFLQNMKS SAEVTPDLQS RGELCELLET TSESNLEDSI AVGPIVPEMA QGEA QWFQE AKNLNEQLRA AYTSASFRHM SLLDISSDLA TDHLLGCDLS IASKHISKPV QEPLVLPEVF GNLNSVMCVE GEAGS GKTV LLKKIAFLWA SGCCPLLNRF QLVFYLSLSS TRPDEGLASI ICDQLLEKEG SVTEMCVRNI IQQLKNQVLF LLDDYK EIC SIPQVIGKLI QKNHLSRTCL LIAVRTNRAR DIRRYLETIL EIKAFPFYNT VCILRKLFSH NMTRLRKFMV YFGKNQS LQ KIQKTPLFVA AICAHWFQYP FDPSFDDVAV FKSYMERLSL RNKATAEILK ATVSSCGELA LKGFFSCCFE FNDDDLAE A GVDEDEDLTM CLMSKFTAQR LRPFYRFLSP AFQEFLAGMR LIELLDSDRQ EHQDLGLYHL KQINSPMMTV SAYNNFLNY VSSLPSTKAG PKIVSHLLHL VDNKESLENI SENDDYLKHQ PEISLQMQLL RGLWQICPQA YFSMVSEHLL VLALKTAYQS NTVAACSPF VLQFLQGRTL TLGALNLQYF FDHPESLSLL RSIHFPIRGN KTSPRAHFSV LETCFDKSQV PTIDQDYASA F EPMNEWER NLAEKEDNVK SYMDMQRRAS PDLSTGYWKL SPKQYKIPCL EVDVNDIDVV GQDMLEILMT VFSASQRIEL HL NHSRGFI ESIRPALELS KASVTKCSIS KLELSAAEQE LLLTLPSLES LEVSGTIQSQ DQIFPNLDKF LCLKELSVDL EGN INVFSV IPEEFPNFHH MEKLLIQISA EYDPSKLVKL IQNSPNLHVF HLKCNFFSDF GSLMTMLVSC KKLTEIKFSD SFFQ AVPFV ASLPNFISLK ILNLEGQQFP DEETSEKFAY ILGSLSNLEE LILPTGDGIY RVAKLIIQQC QQLHCLRVLS FFKTL NDDS VVEIAKVAIS GGFQKLENLK LSINHKITEE GYRNFFQALD NMPNLQELDI SRHFTECIKA QATTVKSLSQ CVLRLP RLI RLNMLSWLLD ADDIALLNVM KERHPQSKYL TILQKWILPF SPIIQK

UniProtKB: Baculoviral IAP repeat-containing protein 1

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Macromolecule #2: NLR family CARD domain-containing protein 4

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 116.941531 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AEYCFNMNFI KDNSRALIQR MGMTVIKQIT DDLFVWNVLN REEVNIICCE KVEQDAARGI IHMILKKGSE SCNLFLKSLK EWNYPLFQD LNGQSLFHQT SEGDLDDLAQ DLKDLYHTPS FLNFYPLGED IDIIFNLKST FTEPVLWRKD QHHHRVEQLT L NGLLQALQ ...String:
AEYCFNMNFI KDNSRALIQR MGMTVIKQIT DDLFVWNVLN REEVNIICCE KVEQDAARGI IHMILKKGSE SCNLFLKSLK EWNYPLFQD LNGQSLFHQT SEGDLDDLAQ DLKDLYHTPS FLNFYPLGED IDIIFNLKST FTEPVLWRKD QHHHRVEQLT L NGLLQALQ SPCIIEGESG KGKSTLLQRI AMLWGSGKCK ALTKFKFVFF LRLSRAQGGL FETLCDQLLD IPGTIRKQTF MA MLLKLRQ RVLFLLDGYN EFKPQNCPEI EALIKENHRF KNMVIVTTTT ECLRHIAQFG ALTAEVGDMT EDSAQALIRE VLI KELAEG LLLQIQKSRC LRNLMKTPLF VVITCAIQMG ESEFHSHTQT TLFHTFYDLL IQKNKHKHKG VAASDFIRSL DHCG DLALE GVFSHKFDFE LQDVSSVNED VLLTTGLLCK YTAQRFKPKY KFFHKSFQEY TAGRRLSSLL TSHEPEEVTK GNGYL QKMV SISDITSTYS SLLRYTCGSS VEATRAVMKH LAAVYQHGCL LGLSIAKRPL WRQESLQSVK NTTEQEILKA ININSF VEC GIHLYQESTS KSALSQEFEA FFQGKSLYIN SGNIPDYLFD FFEHLPNCAS ALDFIKLDFY GGAMASWEKA AEDTGGI HM EEAPETYIPS RAVSLFFNWK QEFRTLEVTL RDFSKLNKQD IRYLGKIFSS ATSLRLQIKR CAGVAGSLSL VLSTCKNI Y SLMVEASPLT IEDERHITSV TNLKTLSIHD LQNQRLPGGL TDSLGNLKNL TKLIMDNIKM NEEDAIKLAE GLKNLKKMC LFHLTHLSDI GEGMDYIVKS LSSEPCDLEE IQLVSCCLSA NAVKILAQNL HNLVKLSILD LSENYLEKDG NEALHELIDR MNVLEQLTA LMLPWGCDVQ GSLSSLLKHL EEVPQLVKLG LKNWRLTDTE IRILGAFFGK NPLKNFQQLN LAGNRVSSDG W LAFMGVFE NLKQLVFFDF STKEFLPDPA LVRKLSQVLS KLTFLQEARL VGWQFDDDDL SVITGAFKLV TA

UniProtKB: NLR family CARD domain-containing protein 4

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Macromolecule #3: Lethal factor,Type III secretion system protein

MacromoleculeName: Lethal factor,Type III secretion system protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: anthrax lethal factor endopeptidase
Source (natural)Organism: Burkholderia (bacteria)
Molecular weightTheoretical: 42.830836 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GSSHHHHHHS SGLVPRGSHM AGGHGDVGMH VKEKEKNKDE NKRKDEERNK TQEEHLKEIM KHIVKIEVKG EEAVKKEAAE KLLEKVPSD VLEMYKAIGG KIYIVDGDIT KHISLEALSE DKKKIKDIYG KDALLHEHYV YAKEGYEPVL VIQSSEDYVE N TEKALNVY ...String:
GSSHHHHHHS SGLVPRGSHM AGGHGDVGMH VKEKEKNKDE NKRKDEERNK TQEEHLKEIM KHIVKIEVKG EEAVKKEAAE KLLEKVPSD VLEMYKAIGG KIYIVDGDIT KHISLEALSE DKKKIKDIYG KDALLHEHYV YAKEGYEPVL VIQSSEDYVE N TEKALNVY YEIGKILSRD ILSKINQPYQ KFLDVLNTIK NASDSDGQDL LFTNQLKEHP TDFSVEFLEQ NSNEVQEVFA KA FAYYIEP QHRDVLQLYA PEAFNYMDKF NEQEINLSLE ELKDQRSTRS NPPTPLLTDY EWSGYLTGIG RAFDTGVKDL NQQ LQDAQA NLTKNPSDPT ALANYQMIMS EYNLYRNAQS SAVKSMKDID SSIVSNFR

UniProtKB: Lethal factor, Type III secretion system protein

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 106381
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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