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

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Basic information

Entry
Database: PDB / ID: 8uaw
TitleCryo-EM Structure of Brucella Abortus Lumazine Synthase (BLS) Engineered with Shiga Toxin II subunit B (Stx2B)
ComponentsShiga toxin II 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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 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 II subunit B (Stx2B)
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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)255,16810
Polymers255,16810
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Shiga toxin II subunit B,6,7-dimethyl-8-ribityllumazine synthase 2 / Stx2 subunit B


Mass: 25516.785 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 bv. 1 str. 9-941 (bacteria)
Gene: stx2B, ribH2 / Plasmid: pET11a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7UQX3, UniProt: P61711
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-Stx2B protein / Type: COMPLEX
Details: Engineered chimera of Brucella abortus Lumazine Synthase (BLS) and Shiga Toxin 2 subunit B (Stx2B)
Entity ID: all / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.234 MDaYES
21NO
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: 42.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3752
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionBlob picking and template picking jobs were 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: 3675008
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96992 / 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
23MXG

3mxg

13MXG2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318250
ELECTRON MICROSCOPYf_angle_d0.46324690
ELECTRON MICROSCOPYf_dihedral_angle_d3.6282450
ELECTRON MICROSCOPYf_chiral_restr0.0432730
ELECTRON MICROSCOPYf_plane_restr0.0033190

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