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- EMDB-48125: Cryo-EM Map of Brucella Abortus Lumazine Synthase (BLS) Engineere... -

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Entry
Database: EMDB / ID: EMD-48125
TitleCryo-EM Map of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin I subunit B (Stx1B) Complexed with Equine Polyclonal Fab Fragments (Complex 3)
Map data
Sample
  • Complex: Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3
KeywordsCHIMERA / SHIGA / TOXIN / IMMUNOGEN / COMPLEX
Biological speciesEscherichia coli O157:H7 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.31 Å
AuthorsCristofalo AE / Sharma A / Cerutti ML / Sharma K / Melero R / Zylberman V / Goldbaum FA / Borgnia MJ / Otero LH
Funding support Argentina, United States, 2 items
OrganizationGrant numberCountry
Agencia Nacional de Promocion Cientifica y Tecnologica (FONCYT)PICT 2020-3047 Argentina
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES103326 United States
CitationJournal: Protein Sci / Year: 2025
Title: Cryo-EM structures of engineered Shiga toxin-based immunogens capable of eliciting neutralizing antibodies with therapeutic potential against hemolytic uremic syndrome.
Authors: Alejandro Ezequiel Cristófalo / Arvind Sharma / María Laura Cerutti / Kedar Sharma / Roberto Melero / Romina Pardo / Fernando Alberto Goldbaum / Mario Borgnia / Vanesa Zylberman / Lisandro Horacio Otero /
Abstract: Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is a serious disease that causes renal failure predominantly in children. Despite its significant impact, there ...Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is a serious disease that causes renal failure predominantly in children. Despite its significant impact, there are currently no licensed vaccines or effective therapies available. The B subunits of Shiga toxins 1 and 2 (Stx1B and Stx2B) are suitable targets for developing neutralizing antibodies, but their pentameric assembly is unstable when isolated from the whole toxin. Taking advantage of the oligomeric symmetry shared between Stx1B and Stx2B with the lumazine synthase from Brucella spp. (BLS), we have previously engineered the chimeric toxoids BLS-Stx1B and BLS-Stx2B as immunogens to generate therapeutic equine polyclonal antibodies. The resulting product (INM004) has successfully passed Phases 1 and 2 clinical trials, and a Phase 3 has been launched in Argentina and seven European countries. In this work, we present the cryo-electron microscopy structures of BLS-Stx1B and BLS-Stx2B, which confirm that these engineered immunogens effectively stabilize the StxB pentamers. Moreover, our results reveal that both chimeric constructs present high flexibility at their extremes, corresponding to motions of the StxBs with respect to the BLS core. Additionally, we present structural evidence of the interaction between the chimeras and polyclonal Fab (pFab) fragments derived from INM004, demonstrating that the elicited neutralizing antibodies block most of the interaction surface of the toxins with their cellular receptors. These findings further validate this promising antibody-based therapy for mitigating STEC-HUS and demonstrate that the BLS-Stx1B and BLS-Stx2B chimeras are potential candidates for developing a human vaccine.
History
DepositionDec 2, 2024-
Header (metadata) releaseOct 22, 2025-
Map releaseOct 22, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48125.png

Downloads & links

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Map

FileDownload / File: emd_48125.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 300 pix.
= 396. Å		1.32 Å/pix.
x 300 pix.
= 396. Å		1.32 Å/pix.
x 300 pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0424
Minimum - Maximum-0.11121285 - 0.34590653
Average (Standard dev.)0.00028517915 (±0.0091545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 396.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_48125_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_48125_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3

EntireName: Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3
Components
  • Complex: Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3

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Supramolecule #1: Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3

SupramoleculeName: Chimeric BLS-Stx1B protein complexed with polyclonal Fab 3
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Engineered chimera of Brucella abortus Lumazine Synthase (BLS) and Shiga Toxin 1 subunit B (Stx1B) in complex with polyclonal Fab fragment from equine antiserum
Source (natural)Organism: Escherichia coli O157:H7 (bacteria) / Location in cell: extracellular region

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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

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
137.0 mMNaClsodium chloride
2.7 mMKClpotassium chloride
10.0 mMNa2HPO4sodium hydrogen phosphate
1.8 mMKH2PO4potassium dihydrogen phosphate
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsPreliminary grid screening was performed using SmartScope software
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6132 / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 317345
CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Software - details: Patch CTF job was used / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab-initio model was generated
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 22151
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Ab-initio job was used
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Software - details: Homogeneous reconstruction only job was used
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)

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