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- EMDB-50637: Structure of the NbNRC2 hexameric resistosome -

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Basic information

Entry
Database: EMDB / ID: EMD-50637
TitleStructure of the NbNRC2 hexameric resistosome
Map dataNRC2 EM map (sharpened)
Sample
  • Complex: NbNRC2a
    • Protein or peptide: NRC2a
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsPlant immunity / complex / IMMUNE SYSTEM
Function / homology
Function and homology information


defense response to other organism / ADP binding
Similarity search - Function
Virus X resistance protein-like, coiled-coil domain / Rx, N-terminal / Rx N-terminal domain / Disease resistance protein Winged helix domain / Disease resistance protein, plants / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / : / Leucine-rich repeat region ...Virus X resistance protein-like, coiled-coil domain / Rx, N-terminal / Rx N-terminal domain / Disease resistance protein Winged helix domain / Disease resistance protein, plants / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / : / Leucine-rich repeat region / Leucine-rich repeat domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesNicotiana benthamiana (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsWebster MW / Madhuprakash J / Kamoun S
Funding support United Kingdom, European Union, 7 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01102X/1 United Kingdom
UK Research and Innovation (UKRI)MR/X033481/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BBSRC BBS/E/J/000PR9795 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BBSRC BBS/E/J/000PR9796 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BBSRC BBS/E/J/000PR9798 United Kingdom
European Research Council (ERC)743165European Union
Gatsby Charitable FoundationGatsby Charitable Foundation United Kingdom
CitationJournal: Sci Adv / Year: 2024
Title: A disease resistance protein triggers oligomerization of its NLR helper into a hexameric resistosome to mediate innate immunity.
Authors: Jogi Madhuprakash / AmirAli Toghani / Mauricio P Contreras / Andres Posbeyikian / Jake Richardson / Jiorgos Kourelis / Tolga O Bozkurt / Michael W Webster / Sophien Kamoun /
Abstract: NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute immune responses triggered by sensor NLRs. The resting state of NbNRC2 was recently shown to be ...NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute immune responses triggered by sensor NLRs. The resting state of NbNRC2 was recently shown to be a homodimer, but the sensor-activated state remains unclear. Using cryo-EM, we determined the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like resistosome. Mutagenesis of the oligomerization interface abolished immune signaling, confirming the functional significance of the NbNRC2 resistosome. Comparative structural analyses between the resting state homodimer and sensor-activated homohexamer revealed substantial rearrangements, providing insights into NLR activation mechanisms. Furthermore, structural comparisons between NbNRC2 hexamer and previously reported CC-NLR pentameric assemblies revealed features allowing an additional protomer integration. Using the NbNRC2 hexamer structure, we assessed the recently released AlphaFold 3 for predicting activated CC-NLR oligomers, revealing high-confidence modeling of NbNRC2 and other CC-NLR amino-terminal α1 helices, a region proven difficult to resolve structurally. Overall, our work sheds light on NLR activation mechanisms and expands understanding of NLR structural diversity.
History
DepositionJun 13, 2024-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50637.png

Downloads & links

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Map

FileDownload / File: emd_50637.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNRC2 EM map (sharpened)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 397.44 Å		0.83 Å/pix.
x 480 pix.
= 397.44 Å		0.83 Å/pix.
x 480 pix.
= 397.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.9588643 - 3.3062196
Average (Standard dev.)0.00034198855 (±0.056026623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 397.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: NRC2 EM map (unsharpened)

Fileemd_50637_additional_1.map
AnnotationNRC2 EM map (unsharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: NRC2 EM map (half map 1)

Fileemd_50637_half_map_1.map
AnnotationNRC2 EM map (half map 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: NRC2 EM map (half map 2)

Fileemd_50637_half_map_2.map
AnnotationNRC2 EM map (half map 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NbNRC2a

EntireName: NbNRC2a
Components
  • Complex: NbNRC2a
    • Protein or peptide: NRC2a
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: NbNRC2a

SupramoleculeName: NbNRC2a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The NbNRC2 was activated in planta to a hexameric resistosome using the sensor Rx and PVX coat protein.
Source (natural)Organism: Nicotiana benthamiana (plant)
Molecular weightTheoretical: 606 KDa

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Macromolecule #1: NRC2a

MacromoleculeName: NRC2a / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Nicotiana benthamiana (plant)
Molecular weightTheoretical: 101.27 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString: MANVAVEFLV QNLMQLLRDN AELIVGVKDS AESLLQDLND FNAFLKQTAK SRTENDVHKE LVKKIKTVVN SAEDAIDKFV IEAKLHKDK GVGRFVDVKH YKRVYDVAGE IKTIRDKVKE IRLNNALDLQ ALQDEDQSAK GVQERKPPVV EEDDVVGFEE E ADKVINRL ...String:
MANVAVEFLV QNLMQLLRDN AELIVGVKDS AESLLQDLND FNAFLKQTAK SRTENDVHKE LVKKIKTVVN SAEDAIDKFV IEAKLHKDK GVGRFVDVKH YKRVYDVAGE IKTIRDKVKE IRLNNALDLQ ALQDEDQSAK GVQERKPPVV EEDDVVGFEE E ADKVINRL LGGSSGLEVV PVVGMPGLGK TTLANKIYKH PDIGYQFFTR IWVYVSQSYR RRELFLNIIS KFTRNTKQYH DM CEEDLAD EIEDFLGKGG KYLIVLDDVW SPDAWERIRI AFPNNNKSNR ILLTTRDSKV AKQCKQCIGI PHDLKFLTED ESW ILLEKK VFHKDKCPPE LELSGKSIAK KCNGLPLAIV VIAGALIGKG KTSREWKQVD ESVGEHLINK DQPENCNKLV QLSY DRLSY DLKACFLYCG AFPGGFEIPA WKLIRLWIAE GFIQYKGHLS LECKAEDNLN DLINRNLVMV MQRTSDGQIK TCRLH DMLH EFCRQEAMKE ENLFQEIKLG AEQYFPGKRE LATYRRLCIH SSVLEFISTK PSGEHVRSFL SFSLKKIEMP SVDIPT IPK GFPLLRVFDV ESINFSRFSK EFFQLYHLRY IAFSSDTIKI IPKHIGELWN IQTLIINTQQ RSLDIQANIW NMERLRH LH TNSSAKLPVP VTPRSSKVPL VNQSLQTLST IAPESCTEEV FARTPNLKKL GIRGKIAVLL EPNKSLLKNV KKLESLEN L KLINDSSQTG KGLRLPPSYI FPTKLRKLSL VDTWLEWNDM SILGQMEHLE VLKLKENGFM GECWESVGGF CSLLVLWIE RTDLVSWKAS ADHFPRLKHL VLICCDKLKE IPIGLADIRS FQVMELQNST KTAAISARGI RDKKDKQTQE GTNNNGFKLS IFPPDL

UniProtKB: NRC2a

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 6 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

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

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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

Concentration2.4 mg/mL
BufferpH: 7.5
Details: 100mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 3% glycerol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 12908 / Average exposure time: 1.95 sec. / Average electron dose: 49.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 391586
CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.1) / Number images used: 205897
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.1)
FSC plot (resolution estimation)

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