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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 1 B subunit
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: To Be Published
Title: Cryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin I subunit B (Stx1B)
Authors: Cristofalo, A.E. / Sharma, A. / Cerutti, M.L. / Sharma, K. / Zylberman, V. / Goldbaum, F.A. / Borgnia, M.J. / Otero, L.H.
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

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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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