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- EMDB-38679: Cryo-EM structure of tomato NRC2 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-38679
TitleCryo-EM structure of tomato NRC2 dimer
Map data
Sample
  • Complex: NRC2 dimer
    • Protein or peptide: NRC2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: INOSITOL HEXAKISPHOSPHATE
Keywordstomato / helper NLR / dimer / PLANT PROTEIN
Function / homology
Function and homology information


defense response to other organism / ADP binding / membrane
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 speciesSolanum lycopersicum (tomato)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsSun Y / Ma SC / Chai JJ
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Nature / Year: 2024
Title: Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.
Authors: Shoucai Ma / Chunpeng An / Aaron W Lawson / Yu Cao / Yue Sun / Eddie Yong Jun Tan / Jinheng Pan / Jan Jirschitzka / Florian Kümmel / Nitika Mukhi / Zhifu Han / Shan Feng / Bin Wu / Paul ...Authors: Shoucai Ma / Chunpeng An / Aaron W Lawson / Yu Cao / Yue Sun / Eddie Yong Jun Tan / Jinheng Pan / Jan Jirschitzka / Florian Kümmel / Nitika Mukhi / Zhifu Han / Shan Feng / Bin Wu / Paul Schulze-Lefert / Jijie Chai /
Abstract: Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors. Maintaining a balanced immune response is crucial, as excessive NLR ...Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP) or pentakisphosphate (IP) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.
History
DepositionJan 13, 2024-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_38679.png

Downloads & links

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Map

FileDownload / File: emd_38679.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.2201469 - 1.6737925
Average (Standard dev.)-0.00021555432 (±0.027167948)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38679_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_38679_half_map_2.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NRC2 dimer

EntireName: NRC2 dimer
Components
  • Complex: NRC2 dimer
    • Protein or peptide: NRC2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: INOSITOL HEXAKISPHOSPHATE

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Supramolecule #1: NRC2 dimer

SupramoleculeName: NRC2 dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Solanum lycopersicum (tomato)

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Macromolecule #1: NRC2

MacromoleculeName: NRC2 / type: protein_or_peptide / ID: 1
Details: Sequence reference for NRC2 is not available in UniProt at the time of biocuration. Current sequence reference is from UniProt id A0A3Q7IF17. Regarding SlNRC2 with the accession number ...Details: Sequence reference for NRC2 is not available in UniProt at the time of biocuration. Current sequence reference is from UniProt id A0A3Q7IF17. Regarding SlNRC2 with the accession number Solyc10g047320 in the SGN database, here is the link: https://solgenomics.net/locus/36701/view. The source article for SlNRC2 is "Helper NLR proteins NRC2a/b and NRC3 but not NRC1 are required for Pto-mediated cell death and resistance in Nicotiana benthamiana" by Wu, Chih-Hang et al. The article was published in The New Phytologist, volume 209, issue 4, in 2016. The DOI is 10.1111/nph.13764.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Solanum lycopersicum (tomato)
Molecular weightTheoretical: 86.267594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DDDLSARGSE ERKPPVVEED DVVGFDEEAD IVINRLLGES NHLEVVPVVG MPGLGKTTLA NKIYKHPKIG YEFFTRIWVY VSQSYRRRE LFLNIISKFT RNTKQYHGMC EEDLADEIQE FLGKGGKYLV VLDDVWSDEA WERIKIAFPN NNKPNRVLLT T RDSKVAKQ ...String:
DDDLSARGSE ERKPPVVEED DVVGFDEEAD IVINRLLGES NHLEVVPVVG MPGLGKTTLA NKIYKHPKIG YEFFTRIWVY VSQSYRRRE LFLNIISKFT RNTKQYHGMC EEDLADEIQE FLGKGGKYLV VLDDVWSDEA WERIKIAFPN NNKPNRVLLT T RDSKVAKQ CNPIPHDLKF LTEDESWILL EKKVFHKDKC PPELVLSGKS IAKKCKGLPL AIVVIAGALI GKGKTPREWK QV DDSVSEH LINRDHPENC NKLVQMSYDR LPYDLKACFL YCSAFPGGFQ IPAWKLIRLW IAEGFIQYKG HLSLECKGED NLN DLINRN LVMVMERTSD GQIKTCRLHD MLHEFCRQEA MKEENLFQEI KLGSEQYFPG KRELSTYRRL CIHSSVLDFF STKP SAEHV RSFLSFSSKK IEMPSADIPT IPKGFPLLRV LDVESINFSR FSREFYQLYH LRYVAFSSDS IKILPKLMGE LWNIQ TIII NTQQRTLDIQ ANIWNMERLR HLHTNSSAKL PVPVAPKNSK VTLVNQSLQT LSTIAPESCT EEVFARTPNL KKLGIR GKI SVLLDNKSAA SLKNVKRLEY LENLKLINDS SIQTSKLRLP PAYIFPTKLR KLTLLDTWLE WKDMSILGQL EHLEVLK MK ENGFSGESWE STGGFCSLLV LWIERTNLVS WKASADDFPR LKHLVLICCD NLKEVPIALA DIRSFQVMML QNSTKTAA I SARQIQAKKD NQTQQGTKNI AFKLSIFPPD L

UniProtKB: NRC1

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: INOSITOL HEXAKISPHOSPHATE

MacromoleculeName: INOSITOL HEXAKISPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: IHP
Molecular weightTheoretical: 660.035 Da
Chemical component information

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 560935
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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