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- PDB-9vcs: Cryo-EM structure of semi-closed state of Botulinum neurotoxin se... -

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Basic information

Entry
Database: PDB / ID: 9vcs
TitleCryo-EM structure of semi-closed state of Botulinum neurotoxin serotype A at pH 7.4
ComponentsBotulinum neurotoxin type A
KeywordsTOXIN / Botulinum neurotoxin serotype A / Cryo-EM
Function / homology
Function and homology information


bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / transmembrane protein transporter activity / metalloendopeptidase activity / toxin activity / proteolysis / extracellular region / zinc ion binding
Similarity search - Function
Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily ...Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / 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 A
Similarity search - Component
Biological speciesClostridium botulinum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsZerang, Z. / Liu, Z. / He, J. / Wang, X. / Qi, F. / Lei, D. / Zhi, D. / Wang, D.
Funding support China, 5items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82073825 China
National Natural Science Foundation of China (NSFC)32171300 China
Other government22ZD6FA053
Other government20JR10RA619
Other governmentlzuyxcx-2022-197
CitationJournal: Adv Sci (Weinh) / Year: 2026
Title: Cryo-EM Structure Guided Engineering of Botulinum Neurotoxin A With Advanced Receptor Binding Affinity and Therapeutical Benefits.
Authors: Wenrui Wang / Zhaxi Zerang / Linjin You / Ziye Liu / Rong Nie / Fuwei Qi / Fenfen Gao / Chengmu Zhao / Wantong Ma / Jinghan He / Xiaoru Wang / Shanquan Wu / Bo Liu / Xinyao Liu / Dongsheng ...Authors: Wenrui Wang / Zhaxi Zerang / Linjin You / Ziye Liu / Rong Nie / Fuwei Qi / Fenfen Gao / Chengmu Zhao / Wantong Ma / Jinghan He / Xiaoru Wang / Shanquan Wu / Bo Liu / Xinyao Liu / Dongsheng Lei / Dejuan Zhi / Dongsheng Wang /
Abstract: Botulinum neurotoxin type A (BoNT/A) has been extensively used in treating a wide range of neurological disorders and aesthetics. However, insufficient binding affinity between BoNT/A and its ...Botulinum neurotoxin type A (BoNT/A) has been extensively used in treating a wide range of neurological disorders and aesthetics. However, insufficient binding affinity between BoNT/A and its receptor SV2C could lead to mild to severe side effects. An open active conformation of BoNT/A cryo-EM structure at a resolution of ∼2.85 Å was resolved. Guided by the BoNT/A cryo-EM structure, saturation mutagenesis libraries were constructed and screened by BACTH system to identify mutants that can boost toxin-SV2C affinity. The engineered toxin binding domain achieved up to approximately six-fold improved affinity in SPR analysis, and the engineered toxins exhibited significantly improved binding capacity, and enhanced SNAP25 cleavage efficacy in cultured neurons. Preclinical animal studies, including MLB, DAS, sweat test, and PET/CT assays, demonstrated that the engineered BoNT/A VLTS has higher potency, lower diffusion, and significantly better safety profiles than the BoNT/A wt, which can reduce side effects and benefit future therapeutic applications.
History
DepositionJun 6, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 22, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Botulinum neurotoxin type A


Theoretical massNumber of molelcules
Total (without water)151,0231
Polymers151,0231
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 Botulinum neurotoxin type A / BoNT/A / Bontoxilysin-A / BOTOX


Mass: 151022.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum (strain Hall / ATCC 3502 / NCTC 13319 / Type A) (bacteria)
Gene: botA, bna, CBO0806, CLC_0862 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DPI1
Has protein modificationN

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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 A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Clostridium botulinum (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.2.1particle selection
2PHENIX1.20.1_4487model refinement
5cryoSPARCv4.2.1CTF correction
10cryoSPARCv4.2.1initial Euler assignment
11cryoSPARCv4.2.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230734 / Symmetry type: POINT
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310370
ELECTRON MICROSCOPYf_angle_d0.514031
ELECTRON MICROSCOPYf_dihedral_angle_d4.251348
ELECTRON MICROSCOPYf_chiral_restr0.0441545
ELECTRON MICROSCOPYf_plane_restr0.0031802

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