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- PDB-6j6i: Reconstitution and structure of a plant NLR resistosome conferrin... -

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Basic information

Entry
Database: PDB / ID: 6j6i
TitleReconstitution and structure of a plant NLR resistosome conferring immunity
Components
  • Disease resistance RPP13-like protein 4
  • Probable serine/threonine-protein kinase PBL2
  • Protein kinase superfamily protein
KeywordsPLANT PROTEIN / resistosome
Function / homologyRx N-terminal domain / Serine/threonine-protein kinase, active site / Protein kinase domain profile. / Serine/Threonine protein kinases active-site signature. / Protein kinases ATP-binding region signature. / Protein tyrosine kinase / NB-ARC domain / Protein kinase domain / Virus X resistance protein-like, coiled-coil domain / Leucine-rich repeat domain superfamily ...Rx N-terminal domain / Serine/threonine-protein kinase, active site / Protein kinase domain profile. / Serine/Threonine protein kinases active-site signature. / Protein kinases ATP-binding region signature. / Protein tyrosine kinase / NB-ARC domain / Protein kinase domain / Virus X resistance protein-like, coiled-coil domain / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Protein kinase, ATP binding site / Protein kinase-like domain superfamily / NB-ARC / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase domain / Tat protein binding / ADP binding / defense response / protein self-association / non-specific serine/threonine protein kinase / defense response to Gram-negative bacterium / protein serine/threonine kinase activity / ATP binding / plasma membrane / nucleus / Probable serine/threonine-protein kinase PBL2 / Disease resistance RPP13-like protein 4 / Protein kinase superfamily protein
Function and homology information
Specimen sourceArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWang, J.Z. / Wang, J. / Hu, M.J. / Wang, H.W. / Zhou, J.M. / Chai, J.J.
Funding supportChina , 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China31421001China
National Natural Science Foundation of China31420103906China
CitationJournal: Science / Year: 2019
Title: Reconstitution and structure of a plant NLR resistosome conferring immunity.
Authors: Jizong Wang / Meijuan Hu / Jia Wang / Jinfeng Qi / Zhifu Han / Guoxun Wang / Yijun Qi / Hong-Wei Wang / Jian-Min Zhou / Jijie Chai /
Abstract: Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly ...Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly understood. We reconstituted an active complex containing the coiled-coil NLR ZAR1, the pseudokinase RKS1, uridylated protein kinase PBL2, and 2'-deoxyadenosine 5'-triphosphate (dATP), demonstrating the oligomerization of the complex during immune activation. The cryo-electron microscopy structure reveals a wheel-like pentameric ZAR1 resistosome. Besides the nucleotide-binding domain, the coiled-coil domain of ZAR1 also contributes to resistosome pentamerization by forming an α-helical barrel that interacts with the leucine-rich repeat and winged-helix domains. Structural remodeling and fold switching during activation release the very N-terminal amphipathic α helix of ZAR1 to form a funnel-shaped structure that is required for the plasma membrane association, cell death triggering, and disease resistance, offering clues to the biochemical function of a plant resistosome.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 15, 2019 / Release: Mar 20, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 20, 2019Structure modelrepositoryInitial release
1.1Apr 17, 2019Structure modelData collection / Database references / Structure summaryaudit_author / citation / citation_author_audit_author.name / _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Probable serine/threonine-protein kinase PBL2
B: Protein kinase superfamily protein
C: Disease resistance RPP13-like protein 4
F: Disease resistance RPP13-like protein 4
G: Disease resistance RPP13-like protein 4
L: Disease resistance RPP13-like protein 4
O: Disease resistance RPP13-like protein 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)575,57914
Polyers572,3197
Non-polymers3,2607
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)32500
ΔGint (kcal/M)-83
Surface area (Å2)187760

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Components

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Protein/peptide , 3 types, 7 molecules ABCFGLO

#1: Protein/peptide Probable serine/threonine-protein kinase PBL2 / PBS1-like protein 2 / Protein kinase 2A


Mass: 46356.430 Da / Num. of mol.: 1 / Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: PBL2, APK2A, KIN1, At1g14370, F14L17.14
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: O49839, non-specific serine/threonine protein kinase
#2: Protein/peptide Protein kinase superfamily protein / Protein kinase-like protein / RKS1


Mass: 40142.375 Da / Num. of mol.: 1 / Source: (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: F15B8.100, Resistance related KinaSe 1, RKS1, At3g57710
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q9SVY5
#3: Protein/peptide
Disease resistance RPP13-like protein 4 / Disease resistance protein ZAR1 / Protein HOPZ-ACTIVATED RESISTANCE 1


Mass: 97163.977 Da / Num. of mol.: 5 / Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: RPP13L4, ZAR1, At3g50950, F18B3.230
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q38834

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Non-polymers , 3 types, 7 molecules

#4: Chemical ChemComp-BQL / [(2~{S})-2-azanyl-3-oxidanylidene-propyl] [(2~{R},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]methyl hydrogen phosphate


Mass: 395.259 Da / Num. of mol.: 1 / Formula: C12H18N3O10P
#5: Chemical ChemComp-BQO / [(2~{R},3~{S})-3-azanyl-4-oxidanylidene-butan-2-yl] [(2~{R},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]methyl hydrogen phosphate


Mass: 409.286 Da / Num. of mol.: 1 / Formula: C13H20N3O10P
#6: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 5 / Formula: C10H16N5O12P3 / Deoxyadenosine triphosphate

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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: resistosome / Type: COMPLEX / Entity ID: 1, 2, 3 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Arabidopsis (plant)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Nominal defocus max: 900 nm / Nominal defocus min: 700 nm / Cs: 0.01 mm / C2 aperture diameter: 50 microns
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.13model refinement
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2092456
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 376858 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 3TL8

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