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- EMDB-25771: CryoEM structure of the (NPR1)2-(TGA3)2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25771
TitleCryoEM structure of the (NPR1)2-(TGA3)2 complex
Map dataComposite map
Sample
  • Complex: (NPR1)2-(TGA3)2 complex
    • Protein or peptide: Regulatory protein NPR1
    • Protein or peptide: Transcription factor TGA3
  • Ligand: ZINC ION
  • Ligand: PALMITIC ACID
KeywordsNPR1 / TGA3 / Plant Immunity / PLANT PROTEIN
Function / homology
Function and homology information


response to herbivore / regulation of salicylic acid mediated signaling pathway / regulation of defense response to bacterium / regulation of systemic acquired resistance / negative regulation of defense response / regulation of jasmonic acid mediated signaling pathway / induced systemic resistance / extracellular ATP signaling / response to insect / systemic acquired resistance, salicylic acid mediated signaling pathway ...response to herbivore / regulation of salicylic acid mediated signaling pathway / regulation of defense response to bacterium / regulation of systemic acquired resistance / negative regulation of defense response / regulation of jasmonic acid mediated signaling pathway / induced systemic resistance / extracellular ATP signaling / response to insect / systemic acquired resistance, salicylic acid mediated signaling pathway / systemic acquired resistance / salicylic acid binding / plant-type hypersensitive response / response to salicylic acid / defense response to fungus / response to bacterium / transcription coregulator activity / response to wounding / RNA polymerase II transcription regulator complex / response to heat / nuclear body / response to hypoxia / transcription cis-regulatory region binding / calmodulin binding / protein ubiquitination / defense response to bacterium / DNA-binding transcription factor activity / DNA-templated transcription / positive regulation of DNA-templated transcription / nucleus / metal ion binding / 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 / Basic-leucine zipper domain superfamily / 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
Regulatory protein NPR1 / Transcription factor TGA3
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWu Q / Zhou Y
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseMar 16, 2022-
Map releaseMar 16, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7tad
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25771.png

Downloads & links

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Map

FileDownload / File: emd_25771.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 409.344 Å		1.07 Å/pix.
x 384 pix.
= 409.344 Å		1.07 Å/pix.
x 384 pix.
= 409.344 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.066 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2 / Movie #1: 10
Minimum - Maximum-86.495339999999999 - 118.735600000000005
Average (Standard dev.)0.00055045245 (±1.0950737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 409.344 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0661.0661.066
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z409.344409.344409.344
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-86.495118.7360.001

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Supplemental data

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Sample components

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Entire : (NPR1)2-(TGA3)2 complex

EntireName: (NPR1)2-(TGA3)2 complex
Components
  • Complex: (NPR1)2-(TGA3)2 complex
    • Protein or peptide: Regulatory protein NPR1
    • Protein or peptide: Transcription factor TGA3
  • Ligand: ZINC ION
  • Ligand: PALMITIC ACID

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Supramolecule #1: (NPR1)2-(TGA3)2 complex

SupramoleculeName: (NPR1)2-(TGA3)2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Regulatory protein NPR1

MacromoleculeName: Regulatory protein NPR1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 67.886797 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPAVPRMDTT IDGFADSYEI SSTSFVATDN TDSSIVYLAA EQVLTGPDVS ALQLLSNSFE SVFDSPDDFY SDAKLVLSDG REVSFHRCV LSARSSFFKS ALAAAKKEKD SNNTAAVKLE LKEIAKDYEV GFDSVVTVLA YVYSSRVRPP PKGVSECADE N CCHVACRP ...String:
GPAVPRMDTT IDGFADSYEI SSTSFVATDN TDSSIVYLAA EQVLTGPDVS ALQLLSNSFE SVFDSPDDFY SDAKLVLSDG REVSFHRCV LSARSSFFKS ALAAAKKEKD SNNTAAVKLE LKEIAKDYEV GFDSVVTVLA YVYSSRVRPP PKGVSECADE N CCHVACRP AVDFMLEVLY LAFIFKIPEL ITLYQRHLLD VVDKVVIEDT LVILKLANIC GKACMKLLDR CKEIIVKSNV DM VSLEKSL PEELVKEIID RRKELGLEVP KVKKHVSNVH KALDSDDIEL VKLLLKEDHT NLDDACALHF AVAYCNVKTA TDL LKLDLA DVNHRNPRGY TVLHVAAMRK EPQLILSLLE KGASASEATL EGRTALMIAK QATMAVECNN IPEQCKHSLK GRLC VEILE QEDKREQIPR DVPPSFAVAA DELKMTLLDL ENRVALAQRL FPTEAQAAME IAEMKGTCEF IVTSLEPDRL TGTKR TSPG VKIAPFRILE EHQSRLKALS KTVELGKRFF PRCSAVLDQI MNCEDLTQLA CGEDDTAEKR LQKKQRYMEI QETLKK AFS EDNLELGNSS LTDSTSSTSK STGGKRSNRK LSHRRRGGWS HPQFER

UniProtKB: Regulatory protein NPR1

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Macromolecule #2: Transcription factor TGA3

MacromoleculeName: Transcription factor TGA3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 36.843457 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPANNDQDED RINDKMKRRL AQNREAARKS RLRKKAHVQQ LEESRLKLSQ LEQELVRARQ QGLCVRNSSD TSYLGPAGNM NSGIAAFEM EYTHWLEEQN RRVSEIRTAL QAHIGDIELK MLVDSCLNHY ANLFRMKADA AKADVFFLMS GMWRTSTERF F QWIGGFRP ...String:
GPANNDQDED RINDKMKRRL AQNREAARKS RLRKKAHVQQ LEESRLKLSQ LEQELVRARQ QGLCVRNSSD TSYLGPAGNM NSGIAAFEM EYTHWLEEQN RRVSEIRTAL QAHIGDIELK MLVDSCLNHY ANLFRMKADA AKADVFFLMS GMWRTSTERF F QWIGGFRP SELLNVVMPY VEPLTDQQLL EVRNLQQSSQ QAEEALSQGL DKLQQGLVES IAIQIKVVES VNHGAPMASA ME NLQALES FVNQADHLRQ QTLQQMSKIL TTRQAARGLL ALGEYFHRLR ALSSLWAARP REHTGGDYKD DDDKSSGYPY DVP DYA

UniProtKB: Transcription factor TGA3

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 4 / Number of copies: 2 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.2 mM salicylic acid.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 240 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 287150
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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