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- EMDB-74329: Cryo-EM structure of the Arabidopsis salicylic acid-bound NPR1-ME... -

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Basic information

Entry
Database: EMDB / ID: EMD-74329
TitleCryo-EM structure of the Arabidopsis salicylic acid-bound NPR1-MED15 complex
Map data
Sample
  • Complex: Ternary complex of dimeric NPR1 with salicylic acid and MED15A KIX domain
    • Protein or peptide: Regulatory protein NPR1
    • Protein or peptide: Mediator of RNA polymerase II transcription subunit 15a
  • Ligand: ZINC ION
  • Ligand: 2-HYDROXYBENZOIC ACID
KeywordsNPR1 / MED15 / salicylic acid / SA receptor / mediator / plant immunity / Arabidopsis / PLANT PROTEIN
Function / homology
Function and homology information


induced systemic resistance / regulation of salicylic acid mediated signaling pathway / regulation of defense response to bacterium / regulation of systemic acquired resistance / extracellular ATP signaling / regulation of jasmonic acid mediated signaling pathway / response to insect / negative regulation of defense response / systemic acquired resistance, salicylic acid mediated signaling pathway / salicylic acid binding ...induced systemic resistance / regulation of salicylic acid mediated signaling pathway / regulation of defense response to bacterium / regulation of systemic acquired resistance / extracellular ATP signaling / regulation of jasmonic acid mediated signaling pathway / response to insect / negative regulation of defense response / systemic acquired resistance, salicylic acid mediated signaling pathway / salicylic acid binding / response to salicylic acid / systemic acquired resistance / plant-type hypersensitive response / positive regulation of fatty acid biosynthetic process / mediator complex / defense response to fungus / response to bacterium / transcription coregulator activity / response to wounding / chromatin DNA binding / response to heat / response to hypoxia / nuclear body / transcription coactivator activity / defense response to bacterium / protein ubiquitination / positive regulation of DNA-templated transcription / DNA-templated transcription / zinc ion binding / nucleus / cytoplasm
Similarity search - Function
Mediator of RNA polymerase II transcription subunit 15a/b/c-like / : / Zinc finger C2HC NPR-type 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 / Mediator complex subunit 15, KIX domain / KIX domain ...Mediator of RNA polymerase II transcription subunit 15a/b/c-like / : / Zinc finger C2HC NPR-type 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 / Mediator complex subunit 15, KIX domain / KIX domain / : / ARC105/Med15 mediator subunit C-terminal domain / Coactivator CBP, KIX domain superfamily / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
Mediator of RNA polymerase II transcription subunit 15a / Regulatory protein NPR1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang S / Zheng N
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: To Be Published
Title: Cryo-EM structure of the Arabidopsis salicylic acid-bound NPR1-MED15 complex
Authors: Zhang S / Zheng N
History
DepositionDec 4, 2025-
Header (metadata) releaseJul 29, 2026-
Map releaseJul 29, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74329.png

Downloads & links

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Map

FileDownload / File: emd_74329.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 331.6 Å		0.83 Å/pix.
x 400 pix.
= 331.6 Å		0.83 Å/pix.
x 400 pix.
= 331.6 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.829 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.165
Minimum - Maximum-1.1547505 - 2.0786994
Average (Standard dev.)0.00003130392 (±0.022096967)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 331.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_74329_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_74329_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_74329_half_map_2.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of dimeric NPR1 with salicylic acid and MED15A KI...

EntireName: Ternary complex of dimeric NPR1 with salicylic acid and MED15A KIX domain
Components
  • Complex: Ternary complex of dimeric NPR1 with salicylic acid and MED15A KIX domain
    • Protein or peptide: Regulatory protein NPR1
    • Protein or peptide: Mediator of RNA polymerase II transcription subunit 15a
  • Ligand: ZINC ION
  • Ligand: 2-HYDROXYBENZOIC ACID

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Supramolecule #1: Ternary complex of dimeric NPR1 with salicylic acid and MED15A KI...

SupramoleculeName: Ternary complex of dimeric NPR1 with salicylic acid and MED15A KIX domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: A near full-length version of NPR1 was co-expressed in E. coli and purified with MED15A KIX domain in presence of SA through gravity affinity purification. Fusion protein tags were cleaved ...Details: A near full-length version of NPR1 was co-expressed in E. coli and purified with MED15A KIX domain in presence of SA through gravity affinity purification. Fusion protein tags were cleaved by TEV enzyme. Purified tag-free NPR1-MED15A KIX-SA complex was cross-linked with BS3, further purified with gel filtration and loaded on ANT grids for Cryo-EM.
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 136.45 KDa

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Macromolecule #1: Regulatory protein NPR1

MacromoleculeName: Regulatory protein NPR1 / type: protein_or_peptide / ID: 1
Details: A near full-length version of NPR1 (amino acids 40-564) was used in this study.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 58.778934 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPDVSALQLL SNSFESVFDS PDDFYSDAKL VLSDGREVSF HRCVLSARSS FFKSALAAAK KEKDSNNTAA VKLELKEIAK DYEVGFDSV VTVLAYVYSS RVRPPPKGVS ECADENCCHV ACRPAVDFML EVLYLAFIFK IPELITLYQR HLLDVVDKVV I EDTLVILK ...String:
GPDVSALQLL SNSFESVFDS PDDFYSDAKL VLSDGREVSF HRCVLSARSS FFKSALAAAK KEKDSNNTAA VKLELKEIAK DYEVGFDSV VTVLAYVYSS RVRPPPKGVS ECADENCCHV ACRPAVDFML EVLYLAFIFK IPELITLYQR HLLDVVDKVV I EDTLVILK LANICGKACM KLLDRCKEII VKSNVDMVSL EKSLPEELVK EIIDRRKELG LEVPKVKKHV SNVHKALDSD DI ELVKLLL KEDHTNLDDA CALHFAVAYC NVKTATDLLK LDLADVNHRN PRGYTVLHVA AMRKEPQLIL SLLEKGASAS EAT LEGRTA LMIAKQATMA VECNNIPEQC KHSLKGRLCV EILEQEDKRE QIPRDVPPSF AVAADELKMT LLDLENRVAL AQRL FPTEA QAAMEIAEMK GTCEFIVTSL EPDRLTGTKR TSPGVKIAPF RILEEHQSRL KALSKTVELG KRFFPRCSAV LDQIM NCED LTQLACGEDD TAEKRLQKKQ RYMEIQETLK KAFSEDNLEL G

UniProtKB: Regulatory protein NPR1

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Macromolecule #2: Mediator of RNA polymerase II transcription subunit 15a

MacromoleculeName: Mediator of RNA polymerase II transcription subunit 15a
type: protein_or_peptide / ID: 2
Details: MED15A KIX domain (aa 17-98) was used in this study.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 9.424906 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDTGDWRTQL PPDSRQKIVN KIMETLKKHL PFSGPEGINE LRRIAARFEE KIFSGALNQT DYLRKISMKM LTMETKSQNA AG

UniProtKB: Mediator of RNA polymerase II transcription subunit 15a

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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: 2-HYDROXYBENZOIC ACID

MacromoleculeName: 2-HYDROXYBENZOIC ACID / type: ligand / ID: 4 / Number of copies: 1 / Formula: SAL
Molecular weightTheoretical: 138.121 Da
Chemical component information

ChemComp-SAL:
2-HYDROXYBENZOIC 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

Concentration3.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
0.5 mMTCEPtris(2-carboxyethyl)phosphine

Details: 150 mM NaCl, 20 mM HEPES pH 7.5, 0.5 mM TCEP, 0.1% DM detergent
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was manually applied to grid and blotted outside of the Vitrobot twice before being applied once and two-side blotted with two layers of blotting papers with blot force 0 for 9 ...Details: Sample was manually applied to grid and blotted outside of the Vitrobot twice before being applied once and two-side blotted with two layers of blotting papers with blot force 0 for 9 seconds within the Vitrobot chamber. Grids used: Amorphous Ni-Titanium Holey Foil Grids from Molecular Dimensions; Gold support grid, Ni-Ti holey foil, 300 mesh, 1.2/1.3 Aperture Pitch.
DetailsThe sample was cross-linked with BS3 and mixed with 0.1% DM detergent prior to being loaded on grids. Due to the presence of the detergent, high concentration of the sample was required to get a desired distribution of particles.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Average exposure time: 7.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: cryosparc ab-initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 773558
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 6
FSC plot (resolution estimation)

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