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- EMDB-25768: Cryo-EM maps of the (TGA3)2-(NPR1)2-(TGA3)2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25768
TitleCryo-EM maps of the (TGA3)2-(NPR1)2-(TGA3)2 complex
Map dataconsensus refinement map
Sample
  • Complex: Complex of (TGA3)2-(NPR1)2-(TGA3)2
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWu Q / Zhou Y / Bartesaghi A / Dong X / Zhou P
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) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25768.png

Downloads & links

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Map

FileDownload / File: emd_25768.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationconsensus refinement map
Voxel sizeX=Y=Z: 1.066 Å
Density
Contour LevelBy AUTHOR: 5.5
Minimum - Maximum-95.136955 - 120.01949
Average (Standard dev.)-4.0332195e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_25768_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local Focused Refinement Map (TGA3 dimer)

Fileemd_25768_additional_1.map
AnnotationLocal Focused Refinement Map (TGA3 dimer)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local Focused Refinement Map (TGA3 dimer)

Fileemd_25768_additional_2.map
AnnotationLocal Focused Refinement Map (TGA3 dimer)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local Focused Refinement Map (NPR1 dimer)

Fileemd_25768_additional_3.map
AnnotationLocal Focused Refinement Map (NPR1 dimer)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of (TGA3)2-(NPR1)2-(TGA3)2

EntireName: Complex of (TGA3)2-(NPR1)2-(TGA3)2
Components
  • Complex: Complex of (TGA3)2-(NPR1)2-(TGA3)2

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Supramolecule #1: Complex of (TGA3)2-(NPR1)2-(TGA3)2

SupramoleculeName: Complex of (TGA3)2-(NPR1)2-(TGA3)2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 pH7.5, 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 - 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 94144
FSC plot (resolution estimation)

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