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- PDB-6j5t: Reconstitution and structure of a plant NLR resistosome conferrin... -

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Basic information

Entry
Database: PDB / ID: 6j5t
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 / homology
Function and homology information


positive regulation of defense response to bacterium / Tat protein binding / response to temperature stimulus / regulation of immune response / ADP binding / defense response / : / kinase activity / defense response to Gram-negative bacterium / cell surface receptor signaling pathway ...positive regulation of defense response to bacterium / Tat protein binding / response to temperature stimulus / regulation of immune response / ADP binding / defense response / : / kinase activity / defense response to Gram-negative bacterium / cell surface receptor signaling pathway / non-specific serine/threonine protein kinase / defense response to bacterium / phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / nucleus / plasma membrane
Similarity search - Function
Receptor-like kinase WAK-like / 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 / Protein tyrosine and serine/threonine kinase ...Receptor-like kinase WAK-like / 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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / URIDINE-5'-MONOPHOSPHATE / Probable serine/threonine-protein kinase PBL2 / Disease resistance RPP13-like protein 4 / Serine/threonine-protein kinase ZRK1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWang, J.Z. / Wang, J. / Hu, M.J. / Wang, H.W. / Zhou, J.M. / Chai, J.J.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China31421001 China
National Natural Science Foundation of China31420103906 China
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.
History
DepositionJan 12, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 20, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Data collection / Database references / Structure summary
Category: audit_author / citation / citation_author
Item: _audit_author.name / _citation.journal_volume ..._audit_author.name / _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.0Apr 12, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Data processing / Database references / Derived calculations / Non-polymer description / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_2 / em_3d_reconstruction / em_ctf_correction / em_entity_assembly / em_image_recording / em_software / entity / entity_name_com / entity_src_gen / pdbx_entity_nonpoly / pdbx_initial_refinement_model / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_peptide_omega / pdbx_validate_torsion / refine / struct_asym / struct_conf / struct_conn / struct_ref / struct_ref_seq / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.group_PDB / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site.occupancy / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_reconstruction.resolution_method / _em_ctf_correction.em_image_processing_id / _em_entity_assembly.entity_id_list / _em_image_recording.avg_electron_dose_per_image / _pdbx_nonpoly_scheme.auth_mon_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.entity_id / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_validate_peptide_omega.auth_seq_id_1 / _pdbx_validate_peptide_omega.auth_seq_id_2 / _pdbx_validate_torsion.auth_seq_id / _struct_asym.entity_id / _struct_conf.beg_auth_seq_id / _struct_conf.end_auth_seq_id

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Structure visualization

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Assembly

Deposited unit
I: Probable serine/threonine-protein kinase PBL2
H: Protein kinase superfamily protein
G: Disease resistance RPP13-like protein 4
A: Probable serine/threonine-protein kinase PBL2
B: Protein kinase superfamily protein
C: Disease resistance RPP13-like protein 4
D: Probable serine/threonine-protein kinase PBL2
E: Protein kinase superfamily protein
F: Disease resistance RPP13-like protein 4
J: Probable serine/threonine-protein kinase PBL2
K: Protein kinase superfamily protein
L: Disease resistance RPP13-like protein 4
M: Probable serine/threonine-protein kinase PBL2
N: Protein kinase superfamily protein
O: Disease resistance RPP13-like protein 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)924,01230
Polymers918,31415
Non-polymers5,69815
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area54140 Å2
ΔGint-221 kcal/mol
Surface area293690 Å2

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Components

#1: Protein
Probable serine/threonine-protein kinase PBL2


Mass: 46356.430 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
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*PLUS, non-specific serine/threonine protein kinase
#2: Protein
Protein kinase superfamily protein / Protein kinase-like protein / RKS1


Mass: 40142.375 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
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
Disease resistance RPP13-like protein 4 / Disease resistance protein ZAR1 / Protein HOPZ-ACTIVATED RESISTANCE 1


Mass: 97163.977 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: RPP13L4, ZAR1, At3g50950, F18B3.230
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q38834
#4: Chemical
ChemComp-U5P / URIDINE-5'-MONOPHOSPHATE / Uridine monophosphate


Mass: 324.181 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C9H13N2O9P
#5: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3

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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: #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 K

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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 X / Nominal defocus max: 900 nm / Nominal defocus min: 700 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder 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
IDName
1CTFFIND
2UCSF
3CHIMERA
4PHENIX
5RELION
6RELION
7RELION
8RELION
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2092456
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.4 Å / Num. of particles: 196707 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 3TL8

3tl8

RefinementHighest resolution: 3.4 Å

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