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- PDB-8c8g: Cryo-EM structure of BoNT/Wo-NTNH complex -

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Basic information

Entry
Database: PDB / ID: 8c8g
TitleCryo-EM structure of BoNT/Wo-NTNH complex
Components
  • Putative botulinum-like toxin Wo
  • Structural protein
KeywordsTOXIN / The complex of botulinum neurotoxin-like protein from Weissella oryzae and its non-toxic non-hemagglutinin partner.
Function / homology
Function and homology information


negative regulation of neurotransmitter secretion / bontoxilysin / metalloendopeptidase activity / proteolysis / zinc ion binding / metal ion binding
Similarity search - Function
Bacterial Ig-like domain (group 3) / Ig-like domain, bacterial type / Botulinum/Tetanus toxin, catalytic chain / Kunitz inhibitor STI-like superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Putative botulinum-like toxin Wo / Ig-like domain-containing protein
Similarity search - Component
Biological speciesWeissella oryzae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsKosenina, S. / Skerlova, J. / Stenmark, P.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Swedish Research Council2022-03681, 2018-03406 Sweden
Cancerfonden20 1287 PjF Sweden
CitationJournal: FEBS J / Year: 2024
Title: The cryo-EM structure of the BoNT/Wo-NTNH complex reveals two immunoglobulin-like domains.
Authors: Sara Košenina / Jana Škerlová / Sicai Zhang / Min Dong / Pål Stenmark /
Abstract: The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, ...The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like (Big) domains on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT-NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.
History
DepositionJan 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative botulinum-like toxin Wo
B: Structural protein


Theoretical massNumber of molelcules
Total (without water)323,9342
Polymers323,9342
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14430 Å2
ΔGint-24 kcal/mol
Surface area90920 Å2

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Components

#1: Protein Putative botulinum-like toxin Wo / BoNT/Wo


Mass: 156964.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Weissella oryzae (bacteria) / Gene: WOSG25_110680 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A069CUU9, bontoxilysin
#2: Protein Structural protein


Mass: 166969.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Weissella oryzae (bacteria) / Gene: WOSG25_110670 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A069CVS9

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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-like protein from Weissella oryzae in complex with its non-toxic non-hemagglutinin partner protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Weissella oryzae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solution
IDSpecimen-IDpH
115.5
225.5
Buffer component
IDConc.NameBuffer-ID
125 mMMES1
2150 mMNaCl1
325 mMMES2
4150 mMNaCl2
Specimen
IDConc. (mg/ml)Experiment-IDEmbedding appliedShadowing appliedStaining appliedVitrification applied
10.71NONONOYES
20.21NONONOYES
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11COPPER300Quantifoil R2/2
22COPPER300Quantifoil R1.2/1.3
Vitrification
IDInstrumentCryogen nameHumidity (%)Specimen-IDChamber temperature (K)Entry-ID
1FEI VITROBOT MARK IVETHANE10012778C8G
2FEI VITROBOT MARK IVETHANE10022778C8G

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1600 nm
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5946

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2EPUimage acquisition
4cryoSPARC2CTF correction
9PHENIXmodel refinement
10cryoSPARC2initial Euler assignment
11cryoSPARC2final Euler assignment
12cryoSPARC2classification
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 635143
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283452 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementResolution: 2.98→2.98 Å / Cor.coef. Fo:Fc: 0.701 / SU B: 17.685 / SU ML: 0.302 / ESU R: 0.483
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.39914 --
obs0.39914 206844 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 65.42 Å2
Refinement stepCycle: 1 / Total: 20363
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.01320779
ELECTRON MICROSCOPYr_bond_other_d00.01619485
ELECTRON MICROSCOPYr_angle_refined_deg1.3571.64128143
ELECTRON MICROSCOPYr_angle_other_deg1.4631.58344968
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.22952528
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.29524.3211097
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.091153705
ELECTRON MICROSCOPYr_dihedral_angle_4_deg14.8271577
ELECTRON MICROSCOPYr_chiral_restr0.0680.22766
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0223602
ELECTRON MICROSCOPYr_gen_planes_other0.0030.024768
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.4866.51410118
ELECTRON MICROSCOPYr_mcbond_other6.4846.51510117
ELECTRON MICROSCOPYr_mcangle_it10.7499.79112644
ELECTRON MICROSCOPYr_mcangle_other10.7489.79112645
ELECTRON MICROSCOPYr_scbond_it7.2497.32110661
ELECTRON MICROSCOPYr_scbond_other7.2477.32110659
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other12.68810.60915499
ELECTRON MICROSCOPYr_long_range_B_refined21.06821.06884951
ELECTRON MICROSCOPYr_long_range_B_other21.06821.06884952
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.155 15298 -
obs--100 %

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