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- EMDB-16475: Cryo-EM structure of BoNT/Wo-NTNH complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16475
TitleCryo-EM structure of BoNT/Wo-NTNH complex
Map data
Sample
  • Complex: Botulinum neurotoxin-like protein from Weissella oryzae in complex with its non-toxic non-hemagglutinin partner protein
    • Protein or peptide: Putative botulinum-like toxin Wo
    • Protein or peptide: Structural protein
KeywordsThe complex of botulinum neurotoxin-like protein from Weissella oryzae and its non-toxic non-hemagglutinin partner. / TOXIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsKosenina S / Skerlova J / Stenmark P
Funding support Sweden, 2 items
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, 2023-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16475.png

Downloads & links

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Map

FileDownload / File: emd_16475.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.124
Minimum - Maximum-0.8563855 - 1.2463069
Average (Standard dev.)0.0006790673 (±0.040499955)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16475_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16475_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Botulinum neurotoxin-like protein from Weissella oryzae in comple...

EntireName: Botulinum neurotoxin-like protein from Weissella oryzae in complex with its non-toxic non-hemagglutinin partner protein
Components
  • Complex: Botulinum neurotoxin-like protein from Weissella oryzae in complex with its non-toxic non-hemagglutinin partner protein
    • Protein or peptide: Putative botulinum-like toxin Wo
    • Protein or peptide: Structural protein

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Supramolecule #1: Botulinum neurotoxin-like protein from Weissella oryzae in comple...

SupramoleculeName: Botulinum neurotoxin-like protein from Weissella oryzae in complex with its non-toxic non-hemagglutinin partner protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Weissella oryzae (bacteria)

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Macromolecule #1: Putative botulinum-like toxin Wo

MacromoleculeName: Putative botulinum-like toxin Wo / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: bontoxilysin
Source (natural)Organism: Weissella oryzae (bacteria)
Molecular weightTheoretical: 156.964484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDVLEMFDVN YESPILESFD STTQSLNDVH VFMSRIQMSA YDADGEGRIE YRNLKLYEIS SGIFISTDRL DTGASGVEDD HEMVDYYSS ARLTREFLGE SLDSQKSDYF EGIKKVFSFY KNKCNESRYI KEFFEEIQFR NICGFPKQAG TSSTDIFDQF N SVDVLLQD ...String:
MDVLEMFDVN YESPILESFD STTQSLNDVH VFMSRIQMSA YDADGEGRIE YRNLKLYEIS SGIFISTDRL DTGASGVEDD HEMVDYYSS ARLTREFLGE SLDSQKSDYF EGIKKVFSFY KNKCNESRYI KEFFEEIQFR NICGFPKQAG TSSTDIFDQF N SVDVLLQD PVTSVWNKKV GSKKANIVII PPATNLPITE ACATAGFQPE GFPKLGSGSF FTVQFDPFFS TRFKAHETDD VA LLDPTLT LLHEMTHGLH FQKGIANPVN RSGETPAWAT TWGRVTGDND AFKETPMEEL LTFNKHTIDD DIEISDHLKS TYI GFLYNG RNEDDPTESV DGVYQNVSSF LNQYRGFEIS SDFQHFIESC YGVKYNQESK KFIVNPRNIK RYVQDGFFID EAKF ARILN IKTRSYYTLM PDNLGVWSYR VDILNRLRET FDEDRGLLSQ ELDFHTALTP VVSENPALEL EVAGMQRMVS LPKIK ASYL PSDIKIKNFT GQKISHDTIL DTNISGIIIS KIKYKSDFVV DESMPRSSLN TTNYNLSPIK GTKFETDIRD KTSVKV TVS EITAPMINHV MKLDNSKVLT ERPSLNEDLE ETFKNTKDVY IPKTTAMMKL KEGADQTLGA VGFAVWSGQI LEDLYNL AQ KKEVSIDQIK DDLMSILPFY CAYKNLSAEK YEQAFANATL DAFLIFATDG GGFAGLGITV GAIAINSMYA KAETMEAY D SMFGKYVDQY QNDIKNFTLN AYVQWENNIL SRLWNESRLA ITGFRNMLKT VKTVMEFDAT NQAYSEEDRK IIKAKCEEI FSEFPMLMQT FAKNSMTANL ENASKIFNDI VWQKIKEELD QYVIDSKKYF LDSLEEAYNN GSISAESYYK YQTEAREKFV SPREVIDLY IAAHDTVVKR KRYIRRYSRK YDLATDFKGN TVHLNGLGEG TQDIQDLYGN YSVYADKKTV STQEGHFDQT I KIAKDTNT INKVVLAVSS NNGKEYALNK DEQYTISFWL RMPVPSSSEE RRIFSYSAVS GVNKEVEELI LQVKNNEFVL AT ANLLRNS EFVIEPRIAL NRWVKITIVN ENTRIKVYQN DNLLGLIKDS SRKKPIAQRG TFKFYNYNVD YQLDDISYYN GTI SQRDIK YTFKEDHGQF VYDHWGERLQ YNKAYYLLSD DNKSAFETVY ETKRLKLKSV PGVDIKYLGM NDRVYGYYGG LQFK LVPLD SKNMNNYVRW GDKFTMQSIE TTNLSLAIIQ DNAYFAPTQL KLISNEGKSE EEIFTFDRNI KLQNAAILVG TGNSK QGPI SAYKRGYSGD LWINGARLDG YVTVVNKSNY SNDEIQEKFK WIFVPKDANW VEGSYPYDVP DYALESGKET AAAKFE RQH MDSSTSAA

UniProtKB: Putative botulinum-like toxin Wo

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Macromolecule #2: Structural protein

MacromoleculeName: Structural protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Weissella oryzae (bacteria)
Molecular weightTheoretical: 166.969734 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSDYKDDDD KSGMDNKLKT ENIRYFRTAA GSEDVLEVKA YEVYPNVWAI PSRYMMEPLQ DLDEVTNPEQ FSIYDKKYLA DIQEQDEFL KSIQAAIEDI KKRTFGLELL TAVSGAVPLP KDTGATNTTL QCIDENGKHT HDVVANVVLW GPGNNLNSNR L ISKSDDDS ...String:
MGSDYKDDDD KSGMDNKLKT ENIRYFRTAA GSEDVLEVKA YEVYPNVWAI PSRYMMEPLQ DLDEVTNPEQ FSIYDKKYLA DIQEQDEFL KSIQAAIEDI KKRTFGLELL TAVSGAVPLP KDTGATNTTL QCIDENGKHT HDVVANVVLW GPGNNLNSNR L ISKSDDDS NGIGSMVELI WNPQILIKNI GTNRIKPATD ELVGLLTKAL FRLYGLGLNK IRYPFYQLDD KKYYSLTAED LI SYGGFSA NVVNLQPYYF LEDQFTKVKE KYESAKKRID DIKVNDEYSQ MLTLKYQFDL YSLFHISTSY IVSTVIPAND KYG GLVSYY TGPNALIDSK TDEKLTSMVK IPLKKIKYSK NQSREYDEYD LTNGEDSTQY FENFTFPKSK HVFVETQPTP ENVF VNLPS EEITKIILPV IPAESDLIKI PFQPATPKSI TTELITTDVP TLGLIFPAVK SKQNLSDIKM TSKLSDALDS DKQTF AFDN TLVDKLSELT SVSDAELFGI IRLIKNELLS VIDNFTTFGD NWSCPRWIDY CFQQVFGSDL KNLIVQGDFE KVFNIS DTL ILPKQLPEDI LQLKPYLFYQ WYAKRYTRIL RLESLFYQIL NEHITLIRSL VSSNNKGQYL QGFMNDLDKI AYNAQYM LS DWTIQLGYYD FKNQVTQVIK TSSMTSEFNI DDLLYDYDTF KLTISQFGAD SINNFTPSQD LKLALNDNNS PILLLGND E IKSNGSITQT DDSLDDETSL LLSKNTSFEG NFSAKYLLSS VGVNFTFKSI ENLNFSVDFM NINIAFSNNF FEITQTGQE TKKYSIAKLF GWNSLVYLIK HSSVEIWDIH SNILLVSHDL TAPQNNIVKA PIKLTNLDNE LILKSFEVFE QDEEANYNDI EQGFKNGII YTAKKMPIIV GEKYALKSSI LDDMGILTSD ENKKYPVFST DVEVESSLNI ILESTTGDKI SVDAGVNIRT I NSNGEENY LGIEDNHLIF VPKEEAELFY LKKAVVEDTI DIFYVVKTLG NMFINVERIS DNIYRLNFKA GILYSTMESD ML VLPAEEA NTAFYIQPIG LASLEVKDSV LGEGNPWLKE DNFLDATDDY GNQIDLSDNR ISVTGSVDTD KVGTYSVVYS YTG IDKTNT EKATITVKLD KSSIKTQDST LQNGKEWVRA DNLVEVIDED GNKVDYSDDR IIQEGDVDIN KAGVYDITFR YRGK FKIIS SSFKVTVIND IWYDSIKNAC KTYLIDYGER INDVKGITFQ NILEATRGKL YGYRVVYDNP HDVINQNPPK DFHFD LIKP FDVKNPSRVH LADYSGYLRL FIISTGKINT DIKVKIYAVL ENKDEIEIFD NHQNDKRHEE IAEIYKSNFD DNNYSA DGK YFISVLFKND VQAVVKDEIY GYEIFYSYFT KFRKDTAFQT DGSKRIFFHD YFNFEVPLDY KDSTFINVIL KNGEKIR IY KFAGYYYYSG HHHHHH

UniProtKB: Ig-like domain-containing protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation #1

Preparation ID1
Concentration0.7 mg/mL
BufferpH: 5.5 / Component:
ConcentrationName
25.0 mMMES
150.0 mMNaCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Sample preparation #2

Preparation ID2
Concentration0.2 mg/mL
BufferpH: 5.5 / Component:
ConcentrationName
25.0 mMMES
150.0 mMNaCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 5946 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 635143
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 283452
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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