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- PDB-7qfq: Cryo-EM structure of Botulinum neurotoxin serotype B -

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Basic information

Entry
Database: PDB / ID: 7qfq
TitleCryo-EM structure of Botulinum neurotoxin serotype B
ComponentsBotulinum neurotoxin type B
KeywordsTOXIN / clostridium botulinum / neurotoxin
Function / homology
Function and homology information


Toxicity of botulinum toxin type B (botB) / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / lipid binding / host cell plasma membrane ...Toxicity of botulinum toxin type B (botB) / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / lipid binding / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / membrane
Similarity search - Function
Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily ...Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Botulinum neurotoxin type B
Similarity search - Component
Biological speciesClostridium botulinum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKosenina, S. / Martinez-Carranza, M. / Davies, J.R. / Masuyer, G. / Stenmark, P.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Swedish Research Council2018-03406 Sweden
Cancerfonden20 1287 PjF Sweden
CitationJournal: Toxins (Basel) / Year: 2021
Title: Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM.
Authors: Sara Košenina / Markel Martínez-Carranza / Jonathan R Davies / Geoffrey Masuyer / Pål Stenmark /
Abstract: Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the ...Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.
History
DepositionDec 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Botulinum neurotoxin type B


Theoretical massNumber of molelcules
Total (without water)153,0221
Polymers153,0221
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area66540 Å2

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Components

#1: Protein Botulinum neurotoxin type B / BoNT/B / Bontoxilysin-B


Mass: 153021.922 Da / Num. of mol.: 1 / Mutation: E231Q, H234Y
Source method: isolated from a genetically manipulated source
Details: catalytically inactive variant of botulinum neurotoxin serotype B [E231Q/H234Y]
Source: (gene. exp.) Clostridium botulinum (bacteria) / Gene: botB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P10844

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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: Botulinum neurotoxin serotype B / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Clostridium botulinum (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM HEPES pH7.5, 50mM NaCl, 0.5mM TCEP
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Pure protein, monodisperse by SEC
Specimen supportGrid material: COPPER / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6317

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.1particle selection
2EPUimage acquisition
4cryoSPARC3.1CTF correction
7PHENIX1.19.2model fitting
8Coot0.9.5model fitting
10PHENIX1.19.2model refinement
11cryoSPARC3.1initial Euler assignment
12cryoSPARC3.1final Euler assignment
13cryoSPARC3.1classification
14cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2432309
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286802 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210894
ELECTRON MICROSCOPYf_angle_d0.50514718
ELECTRON MICROSCOPYf_dihedral_angle_d4.2081412
ELECTRON MICROSCOPYf_chiral_restr0.0441585
ELECTRON MICROSCOPYf_plane_restr0.0041894

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