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- EMDB-26565: Apo state Shiga toxin 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-26565
TitleApo state Shiga toxin 2
Map data
Sample
  • Complex: Ternary complex of stx2 and DARPin
    • Complex: Shiga-like toxin 2 subunit A
      • Complex: Shiga-like toxin 2 subunit B
        • Complex: DARPin
Biological speciesEscherichia phage 933W (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsJiang M / Zhang J
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationA1863 United States
CitationJournal: Bioengineering (Basel) / Year: 2022
Title: A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.
Authors: Yu Zeng / Mengqiu Jiang / Sally Robinson / Zeyu Peng / Vikas Chonira / Rudo Simeon / Saul Tzipori / Junjie Zhang / Zhilei Chen /
Abstract: Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC ...Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC) of 0.61 nM The two monomeric DARPin constituents of SD5 exhibit complementary functions-SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin's catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.
History
DepositionMar 30, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26565.png

Downloads & links

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Map

FileDownload / File: emd_26565.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-2.1557276 - 3.5410442
Average (Standard dev.)0.00081771536 (±0.1050095)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26565_half_map_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_26565_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of stx2 and DARPin

EntireName: Ternary complex of stx2 and DARPin
Components
  • Complex: Ternary complex of stx2 and DARPin
    • Complex: Shiga-like toxin 2 subunit A
      • Complex: Shiga-like toxin 2 subunit B
        • Complex: DARPin

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Supramolecule #1: Ternary complex of stx2 and DARPin

SupramoleculeName: Ternary complex of stx2 and DARPin / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia phage 933W (virus)

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Supramolecule #2: Shiga-like toxin 2 subunit A

SupramoleculeName: Shiga-like toxin 2 subunit A / type: complex / ID: 2 / Chimera: Yes / Parent: 1

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Supramolecule #3: Shiga-like toxin 2 subunit B

SupramoleculeName: Shiga-like toxin 2 subunit B / type: complex / ID: 3 / Chimera: Yes / Parent: 2

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Supramolecule #4: DARPin

SupramoleculeName: DARPin / type: complex / ID: 4 / Chimera: Yes / Parent: 3

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1r4p

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142655
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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