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- PDB-7tad: CryoEM structure of the (NPR1)2-(TGA3)2 complex -

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Basic information

Entry
Database: PDB / ID: 7tad
TitleCryoEM structure of the (NPR1)2-(TGA3)2 complex
Components
  • Regulatory protein NPR1
  • Transcription factor TGA3
KeywordsPLANT PROTEIN / NPR1 / TGA3 / Plant Immunity
Function / homology
Function and homology information


response to herbivore / regulation of salicylic acid mediated signaling pathway / regulation of systemic acquired resistance / negative regulation of defense response / response to insect / regulation of jasmonic acid mediated signaling pathway / induced systemic resistance / extracellular ATP signaling / systemic acquired resistance / systemic acquired resistance, salicylic acid mediated signaling pathway ...response to herbivore / regulation of salicylic acid mediated signaling pathway / regulation of systemic acquired resistance / negative regulation of defense response / response to insect / regulation of jasmonic acid mediated signaling pathway / induced systemic resistance / extracellular ATP signaling / systemic acquired resistance / systemic acquired resistance, salicylic acid mediated signaling pathway / plant-type hypersensitive response / defense response to fungus / response to bacterium / response to wounding / RNA polymerase II transcription regulator complex / response to heat / response to hypoxia / protein ubiquitination / calmodulin binding / transcription cis-regulatory region binding / defense response to bacterium / DNA-binding transcription factor activity / DNA-templated transcription / positive regulation of DNA-templated transcription / nucleus / cytoplasm
Similarity search - Function
Transcription factor TGA like domain / Seed dormancy control / DOG1 domain profile. / NPR1/NIM1-like, C-terminal / Regulatory protein NPR, central domain / Regulatory protein NPR / Domain of unknown function (DUF3420) / NPR1/NIM1 like defence protein C terminal / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. ...Transcription factor TGA like domain / Seed dormancy control / DOG1 domain profile. / NPR1/NIM1-like, C-terminal / Regulatory protein NPR, central domain / Regulatory protein NPR / Domain of unknown function (DUF3420) / NPR1/NIM1 like defence protein C terminal / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
PALMITIC ACID / Regulatory protein NPR1 / Transcription factor TGA3
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWu, Q. / Zhou, Y. / Bartesaghi, A. / Dong, X. / Zhou, P.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: Structural basis of NPR1 in activating plant immunity.
Authors: Shivesh Kumar / Raul Zavaliev / Qinglin Wu / Ye Zhou / Jie Cheng / Lucas Dillard / Jordan Powers / John Withers / Jinshi Zhao / Ziqiang Guan / Mario J Borgnia / Alberto Bartesaghi / Xinnian Dong / Pei Zhou /
Abstract: NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid. Despite the important role of NPR1 in plant immunity, understanding of its regulatory ...NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid. Despite the important role of NPR1 in plant immunity, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA3-NPR1-TGA3 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.
History
DepositionDec 20, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release
Revision 1.1May 25, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 28, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Regulatory protein NPR1
B: Regulatory protein NPR1
C: Transcription factor TGA3
D: Transcription factor TGA3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)210,1048
Polymers209,4614
Non-polymers6444
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Regulatory protein NPR1 / BTB/POZ domain-containing protein NPR1 / Non-inducible immunity protein 1 / Nim1 / Nonexpresser of ...BTB/POZ domain-containing protein NPR1 / Non-inducible immunity protein 1 / Nim1 / Nonexpresser of PR genes 1 / Salicylic acid insensitive 1 / Sai1


Mass: 67886.797 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: NPR1, NIM1, SAI1, At1g64280, F15H21.6 / Production host: Escherichia coli (E. coli) / References: UniProt: P93002
#2: Protein Transcription factor TGA3 / bZIP transcription factor 22 / AtbZIP22


Mass: 36843.457 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: TGA3, BZIP22, At1g22070, F2E2.14 / Production host: Escherichia coli (E. coli) / References: UniProt: Q39234
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
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: (NPR1)2-(TGA3)2 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.2 mM salicylic acid.
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277.15 K
Details: 3 ul of the sample was applied to the grid and incubated for 60 s in the chamber set at 277.15 K and 85% humidity. The grid was blotted for 2.4 s, followed by plunge-freezing in liquid ...Details: 3 ul of the sample was applied to the grid and incubated for 60 s in the chamber set at 277.15 K and 85% humidity. The grid was blotted for 2.4 s, followed by plunge-freezing in liquid ethane cooled by liquid nitrogen.

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 287150 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.03 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00288674
ELECTRON MICROSCOPYf_angle_d0.689911732
ELECTRON MICROSCOPYf_chiral_restr0.04091398
ELECTRON MICROSCOPYf_plane_restr0.00661498
ELECTRON MICROSCOPYf_dihedral_angle_d5.03021210

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