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- EMDB-19121: CryoEM structure of the plant helper NLR NRC2 in its resting state -

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Basic information

Entry
Database: EMDB / ID: EMD-19121
TitleCryoEM structure of the plant helper NLR NRC2 in its resting state
Map dataPlease use contour for 0.0034 for atom inclusion. This covers all the main chain atoms in the model
Sample
  • Complex: NRC2 heler NLR dimer with ADP
    • Protein or peptide: NRC2a
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsNLR protein / Helper NLRl / plant immunity / R-protein / IMMUNE SYSTEM
Function / homology
Function and homology information


response to other organism / ADP binding / defense response
Similarity search - Function
Virus X resistance protein-like, coiled-coil domain / Rx, N-terminal / Rx N-terminal domain / Disease resistance protein, plants / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / 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: 3.9 Å
AuthorsSelvaraj M / Kamoun S / Contreras MP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)V002937/1 United Kingdom
Citation
Journal: PLoS Biol / Year: 2024
Title: Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome.
Authors: Muniyandi Selvaraj / AmirAli Toghani / Hsuan Pai / Yu Sugihara / Jiorgos Kourelis / Enoch Lok Him Yuen / Tarhan Ibrahim / He Zhao / Rongrong Xie / Abbas Maqbool / Juan Carlos De la ...Authors: Muniyandi Selvaraj / AmirAli Toghani / Hsuan Pai / Yu Sugihara / Jiorgos Kourelis / Enoch Lok Him Yuen / Tarhan Ibrahim / He Zhao / Rongrong Xie / Abbas Maqbool / Juan Carlos De la Concepcion / Mark J Banfield / Lida Derevnina / Benjamin Petre / David M Lawson / Tolga O Bozkurt / Chih-Hang Wu / Sophien Kamoun / Mauricio P Contreras /
Abstract: Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the ...Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 from Nicotiana benthamiana accumulates in vivo as a homodimer that converts into a higher-order oligomer upon activation by its upstream virus disease resistance protein Rx. The cryo-EM structure of NbNRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation and contribute to immune receptor autoinhibition. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways, possibly to minimise undesired cross-activation and evade pathogen suppression of immunity. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.
#1: Journal: Biorxiv / Year: 2023
Title: Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome
Authors: Selvaraj M / Kamoun S / Contreras MP
History
DepositionDec 12, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19121.png

Downloads & links

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Map

FileDownload / File: emd_19121.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPlease use contour for 0.0034 for atom inclusion. This covers all the main chain atoms in the model
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 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: 0.0034
Minimum - Maximum-0.012051321 - 0.022530898
Average (Standard dev.)0.000101284684 (±0.0007619272)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19121_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19121_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_19121_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NRC2 heler NLR dimer with ADP

EntireName: NRC2 heler NLR dimer with ADP
Components
  • Complex: NRC2 heler NLR dimer with ADP
    • Protein or peptide: NRC2a
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: NRC2 heler NLR dimer with ADP

SupramoleculeName: NRC2 heler NLR dimer with ADP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: This is helper NLR protein NRC2 in inactive state with ADP nucleotide bound state
Source (natural)Organism: Nicotiana benthamiana (plant)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: NRC2a

MacromoleculeName: NRC2a / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Nicotiana benthamiana (plant) / Tissue: leaves
Molecular weightTheoretical: 107.522398 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 IFPPDLGSG SGRGSHHHHH HDYDIPTTAS ENLYFQGELD YKDHDGDYKD HDIDYKDDDD K

UniProtKB: NRC2a

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
100.0 mMTris HCl
150.0 mMNaCl
1.0 mMMagnesium chloride
1.0 mMEDTA
5.0 PercentageGlycerol
GridModel: C-flat / Material: COPPER / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Details: The grids were negatively glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: The C-flat grids were coated with grphene oxide, in house and the sample was applied two times inside the vitrobot chamber.
Detailsthe particles are NRC2 purified from plant after over expression. This was suspended in buffer and cryogrids were made on graphene oxide backing

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6000 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 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: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Tbhe autopick program picked so many particles including non-protein stuffs
Startup modelType of model: OTHER / Details: Abinitio model from Relion
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 229347
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 3 / Avg.num./class: 200000 / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Residue range: 1-880 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: This is the full length sequence of NRC
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 100 / Target criteria: CC
Output model

PDB-8rfh:
CryoEM structure of the plant helper NLR NRC2 in its resting state

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