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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
CitationJournal: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
Water0
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 / Adenosine 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

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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 mMNaClSodium chloride1
31 mMMagnesium chloride1
41 mMEDTAEthylenediaminetetraacetic acid1
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 FIELDBright-field microscopy / 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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