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

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Basic information

Entry
Database: PDB / ID: 8rfh
TitleCryoEM structure of the plant helper NLR NRC2 in its resting state
ComponentsNRC2a
KeywordsIMMUNE SYSTEM / NLR protein / Helper NLRl / plant immunity / R-protein
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
ADENOSINE-5'-DIPHOSPHATE / NRC2a
Similarity search - Component
Biological speciesNicotiana benthamiana (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSelvaraj, M. / Kamoun, S. / Contreras, M.P.
Funding support United Kingdom, 1items
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, M.P.
History
DepositionDec 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NRC2a
B: NRC2a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,8994
Polymers215,0452
Non-polymers8542
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein NRC2a


Mass: 107522.398 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nicotiana benthamiana (plant) / Tissue: leaves / Plasmid: NbNRC2a-HF
Details (production host): This plasmid is compatible with agrobacterium tumifacians carrying NRC2 gene with 3xflag tag
Production host: Nicotiana benthamiana (plant) / References: UniProt: A0A0S3ANR1
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: NRC2 heler NLR dimer with ADP / Type: COMPLEX
Details: This is helper NLR protein NRC2 in inactive state with ADP nucleotide bound state
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Nicotiana benthamiana (plant)
Source (recombinant)Organism: Nicotiana benthamiana (plant)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
1100 mMTris HCl1
2150 mMNaCl1
31 mMMagnesium chloride1
41 mMEDTA1
55 PercentageGlycerol1
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: the particles are NRC2 purified from plant after over expression. This was suspended in buffer and cryogrids were made on graphene oxide backing
Specimen supportDetails: The grids were negatively glow discharged / Grid material: COPPER / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: The C-flat grids were coated with grphene oxide, in house and the sample was applied two times inside the vitrobot chamber

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6000

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4RELIONCTF correction
7Coot0.9.6model fitting
8UCSF ChimeraX1.3model fitting
10RELIONinitial Euler assignment
12RELION4classification
13RELION3D reconstruction
14PHENIX1.20.1_4487:model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionDetails: Tbhe autopick program picked so many particles including non-protein stuffs
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229347 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
Atomic model buildingChain residue range: 1-880 / Details: This is the full length sequence of NRC / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212054
ELECTRON MICROSCOPYf_angle_d0.75816280
ELECTRON MICROSCOPYf_dihedral_angle_d6.721578
ELECTRON MICROSCOPYf_chiral_restr0.0441812
ELECTRON MICROSCOPYf_plane_restr0.0042058

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