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- PDB-8uav: Cryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Eng... -

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Basic information

Entry
Database: PDB / ID: 8uav
TitleCryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin I subunit B (Stx1B)
ComponentsShiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
KeywordsTOXIN / CHIMERA / SHIGA / 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
6,7-dimethyl-8-ribityllumazine synthase 2 / Shiga toxin subunit B
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Brucella abortus biovar 1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsCristofalo, A.E. / Sharma, A. / Cerutti, M.L. / Sharma, K. / Zylberman, V. / Goldbaum, F.A. / Borgnia, M.J. / Otero, L.H.
Funding support Argentina, United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
B: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
C: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
D: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
E: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
F: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
G: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
H: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
I: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2
J: Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2


Theoretical massNumber of molelcules
Total (without water)253,90810
Polymers253,90810
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Shiga toxin subunit B,6,7-dimethyl-8-ribityllumazine synthase 2 / Shiga toxin 1 subunit B / DMRL synthase 2 / LS 2 / Lumazine synthase 2 / BLS / Type II lumazine synthase


Mass: 25390.830 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria), (gene. exp.) Brucella abortus biovar 1 (strain 9-941)
Gene: stx1, ribH2 / Plasmid: pET11a / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q7DH26, UniProt: P61711, 6,7-dimethyl-8-ribityllumazine synthase
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Chimeric BLS-Stx1B protein / Type: COMPLEX
Details: Engineered chimera of Brucella abortus Lumazine Synthase (BLS) and Shiga Toxin 1 subunit B (Stx1B)
Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Escherichia coli O157:H7 (bacteria) / Cellular location: extracellular region
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET11a
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1137 mMsodium chlorideNaCl1
22.7 mMpotassium chlorideKCl1
310 mMsodium hydrogen phosphateNa2HPO41
41.8 mMpotassium dihydrogen phosphateKH2PO41
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Preliminary grid screening was performed using SmartScope software
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5884
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionBlob picking job was used
2Topaz0.2.4particle selection
3EPUimage acquisition
5cryoSPARC4.2.1CTF correctionPatch CTF job was used
8Coot0.9.6model fitting
9UCSF ChimeraX1.3model fitting
11cryoSPARC4.2.1initial Euler assignmentAb-initio job was used
12cryoSPARC4.2.1final Euler assignmentHomogeneous refinement job was used
13cryoSPARC4.2.1classification
14cryoSPARC4.2.13D reconstruction
15PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1773142
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88636 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11XN1

1xn1

11XN11PDBexperimental model
21QNU

1qnu

11QNU2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00218150
ELECTRON MICROSCOPYf_angle_d0.44424580
ELECTRON MICROSCOPYf_dihedral_angle_d5.6892450
ELECTRON MICROSCOPYf_chiral_restr0.0422780
ELECTRON MICROSCOPYf_plane_restr0.0033160

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