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- EMDB-42073: Cryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Eng... -

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Entry
Database: EMDB / ID: EMD-42073
TitleCryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin II subunit B (Stx2B)
Map dataStructure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin II subunit B (Stx2B)
Sample
  • Complex: Chimeric BLS-Stx2B protein
    • Protein or peptide: Shiga toxin II subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
KeywordsCHIMERA / SHIGA / TOXIN / IMMUNOGEN
Function / homology
Function and homology information


symbiont-mediated hemolysis of host erythrocyte / 6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / toxin activity / extracellular region / cytosol
Similarity search - Function
Shiga-like toxin, beta subunit / Shiga-like toxin beta subunit / Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase / Enterotoxin
Similarity search - Domain/homology
Shiga toxin 2 subunit B / 6,7-dimethyl-8-ribityllumazine synthase 2
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria) / Brucella abortus bv. 1 str. 9-941 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsCristofalo AE / Sharma A / Cerutti ML / Sharma K / 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
DepositionSep 22, 2023-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42073.png

Downloads & links

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Map

FileDownload / File: emd_42073.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin II subunit B (Stx2B)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 240 pix.
= 259.2 Å		1.08 Å/pix.
x 240 pix.
= 259.2 Å		1.08 Å/pix.
x 240 pix.
= 259.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.76
Minimum - Maximum-17.497710999999999 - 31.773036999999999
Average (Standard dev.)0.000000000001366 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_42073_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_42073_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Chimeric BLS-Stx2B protein

EntireName: Chimeric BLS-Stx2B protein
Components
  • Complex: Chimeric BLS-Stx2B protein
    • Protein or peptide: Shiga toxin II subunit B,6,7-dimethyl-8-ribityllumazine synthase 2

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Supramolecule #1: Chimeric BLS-Stx2B protein

SupramoleculeName: Chimeric BLS-Stx2B protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Engineered chimera of Brucella abortus Lumazine Synthase (BLS) and Shiga Toxin 2 subunit B (Stx2B)
Source (natural)Organism: Escherichia coli O157:H7 (bacteria) / Location in cell: extracellular region

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Macromolecule #1: Shiga toxin II subunit B,6,7-dimethyl-8-ribityllumazine synthase 2

MacromoleculeName: Shiga toxin II subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Brucella abortus bv. 1 str. 9-941 (bacteria)
Molecular weightTheoretical: 25.516785 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHADCAKGKI EFSKYNEDDT FTVKVDGKEY WTSRWNLQPL LQSAQLTGMT VTIKSSTCES GSGFAEVQFN NDGSGSGSGS GSLKTSFKI AFIQARWHAD IVDEARKSFV AELAAKTGGS VEVEIFDVPG AYEIPLHAKT LARTGRYAAI VGAAFVIDGG I YRHDFVAT ...String:
MHADCAKGKI EFSKYNEDDT FTVKVDGKEY WTSRWNLQPL LQSAQLTGMT VTIKSSTCES GSGFAEVQFN NDGSGSGSGS GSLKTSFKI AFIQARWHAD IVDEARKSFV AELAAKTGGS VEVEIFDVPG AYEIPLHAKT LARTGRYAAI VGAAFVIDGG I YRHDFVAT AVINGMMQVQ LETEVPVLSV VLTPHHFHES KEHHDFFHAH FKVKGVEAAH AALQIVSERS RIAALV

UniProtKB: Shiga toxin 2 subunit B, 6,7-dimethyl-8-ribityllumazine synthase 2

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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

Concentration1.4 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: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN 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: 3752 / Average electron dose: 42.7 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: 3675008
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 reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 96992
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 refinement job was used
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)

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Atomic model buiding 1

Initial model
PDB IDChain

1xn1

source_name: PDB, initial_model_type: experimental model

3mxg

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8uaw:
Cryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin II subunit B (Stx2B)

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