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- EMDB-43786: CryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botu... -

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Basic information

Entry
Database: EMDB / ID: EMD-43786
TitleCryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botulinum strain A1-ST7B, major class
Map data
Sample
  • Complex: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class
    • Protein or peptide: Botulinum neurotoxin type A
    • Protein or peptide: NtnH
    • Protein or peptide: OrfX2
KeywordsBotulinum neurotoxin / Progenitor toxin complex (PTC) / TOXIN
Function / homology
Function and homology information


bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region ...bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region / zinc ion binding / membrane
Similarity search - Function
Clostridium P47 protein / Clostridium P-47-like / Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding ...Clostridium P47 protein / Clostridium P-47-like / Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / 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
OrfX2 / NtnH / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesClostridium botulinum A (bacteria) / Clostridium botulinum A str. Hall (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsGao L
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI139087 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI158503 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI163178 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Botulinum neurotoxins exploit host digestive proteases to boost their oral toxicity via activating OrfXs/P47.
Authors: Linfeng Gao / Maria Barbara Nowakowska / Katja Selby / Adina Przykopanski / Baohua Chen / Maren Krüger / François Paul Douillard / Kwok-Ho Lam / Peng Chen / Ting Huang / Nigel Peter Minton ...Authors: Linfeng Gao / Maria Barbara Nowakowska / Katja Selby / Adina Przykopanski / Baohua Chen / Maren Krüger / François Paul Douillard / Kwok-Ho Lam / Peng Chen / Ting Huang / Nigel Peter Minton / Martin Bernhard Dorner / Brigitte Gertrud Dorner / Andreas Rummel / Miia Lindström / Rongsheng Jin /
Abstract: Botulinum neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 ...Botulinum neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 and P47). The orfX gene cluster is also found in the genomes of many non-BoNT-producing bacteria, often alongside genes encoding oral insecticidal toxins. However, the functions of these OrfXs and P47 remain elusive. Here, we demonstrate that the combined action of all four components (OrfXs and P47) drastically boosts the oral toxicity of BoNT in mice, following proteolytic activation by digestive proteases that oral toxins regularly confront. In particular, OrfX2 adopts a self-inhibiting state, engaging with BoNT through another clostridial protein, nontoxic non-hemagglutinin (NTNH), only after proteolytic activation. Cryo-electron microscopy studies unveil that two molecules of protease-activated OrfX2 simultaneously associate with NTNH, a binding mode crucial for boosting BoNT oral toxicity. Collectively, these studies offer novel insights into the physiological functions and regulatory mechanisms of OrfXs and P47 of BoNTs, shedding light on the pathogenesis of other bacterial toxins associated with homologous OrfXs and P47.
History
DepositionFeb 23, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43786.png

Downloads & links

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Map

FileDownload / File: emd_43786.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 512 pix.
= 403.456 Å		0.79 Å/pix.
x 512 pix.
= 403.456 Å		0.79 Å/pix.
x 512 pix.
= 403.456 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.788 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.22310318 - 0.40663928
Average (Standard dev.)0.000018535313 (±0.00770362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 403.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43786_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43786_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class

EntireName: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class
Components
  • Complex: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class
    • Protein or peptide: Botulinum neurotoxin type A
    • Protein or peptide: NtnH
    • Protein or peptide: OrfX2

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Supramolecule #1: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class

SupramoleculeName: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridium botulinum A (bacteria)
Molecular weightTheoretical: 352 KDa

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Macromolecule #1: Botulinum neurotoxin type A

MacromoleculeName: Botulinum neurotoxin type A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum A str. Hall (bacteria)
Molecular weightTheoretical: 149.479859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLK GVTKLFERIY STDLGRMLLT SIVRGIPFWG GSTIDTELKV IDTNCINVIQ PDGSYRSEEL NLVIIGPSAD I IQFECKSF ...String:
MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLK GVTKLFERIY STDLGRMLLT SIVRGIPFWG GSTIDTELKV IDTNCINVIQ PDGSYRSEEL NLVIIGPSAD I IQFECKSF GHEVLNLTRN GYGSTQYIRF SPDFTFGFEE SLEVDTNPLL GAGKFATDPA VTLAHQLIHA GHRLYGIAIN PN RVFKVNT NAYYEMSGLE VSFEELRTFG GHDAKFIDSL QENEFRLYYY NKFKDIASTL NKAKSIVGTT ASLQYMKNVF KEK YLLSED TSGKFSVDKL KFDKLYKMLT EIYTEDNFVK FFKVLNAKTF LNFDKAVFKI NIVPKVNYTI YDGFNLRNTN LAAN FNGQN TEINNMNFTK LKNFTGLFEF YKLLCVRGII TSKTKSLDKG YNKALNDLCI KVNNWDLFFS PSEDNFTNDL NKGEE ITSD TNIEAAEENI SLDLIQQYYL TFNFDNEPEN ISIENLSSDI IGQLELMPNI ERFPNGKKYE LDKYTMFHYL RAQEFE HGK SRIALTNSVN EALLNPSRVY TFFSSDYVKK VNKATEAAMF LGWVEQLVYD FTDETSEVST TDKIADITII IPYIGPA LN IGNMLYKDDF VGALIFSGAV ILLEFIPEIA IPVLGTFALV SYIANKVLTV QTIDNALSKR NEKWDEVYKY IVTNWLAK V NTQIDLIRKK MKEALENQAE ATKAIINYQY NQYTEEEKNN INFNIDDLSS KLNESINKAM ININKFLNQC SVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLI DLSRYASKIN IGSKVNFDPI DKNQIQLFNL ESSKIEVILK NAIVYNSMYE NFSTSFWIRI PKYFNSISLN N EYTIINCM ENNSGWKVSL NYGEIIWTLQ DTQEIKQRVV FKYSQMINIS DYINRWIFVT ITNNRLNNSK IYINGRLIDQ KP ISNLGNI HASNNIMFKL DGCRDTHRYI WIKYFNLFDK ELNEKEIKDL YDNQSNSGIL KDFWGDYLQY DKPYYMLNLY DPN KYVDVN NVGIRGYMYL KGPRGSVMTT NIYLNSSLYR GTKFIIKKYA SGNKDNIVRN NDRVYINVVV KNKEYRLATN ASQA GVEKI LSALEIPDVG NLSQVVVMKS KNDQGITNKC KMNLQDNNGN DIGFIGFHQF NNIAKLVASN WYNRQIERSS RTLGC SWEF IPVDDGWGER PL

UniProtKB: Botulinum neurotoxin type A

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Macromolecule #2: NtnH

MacromoleculeName: NtnH / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum A (bacteria)
Molecular weightTheoretical: 135.28575 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKINNNFNID SLIDNRDVAI VRGRKTDTFF KVFQVAPNIW VAPERYYGES LNINEDQKSD GGIYDSNFLL TNDEKDEFLQ ATVKILQRI NNNVIGAKLL SLISTAIPFP YEYKPGDYRQ TNYLVSKDNQ HYYTANLVIF GPGTNIVENN AVYYKKEDSE N GMGTMSEI ...String:
MKINNNFNID SLIDNRDVAI VRGRKTDTFF KVFQVAPNIW VAPERYYGES LNINEDQKSD GGIYDSNFLL TNDEKDEFLQ ATVKILQRI NNNVIGAKLL SLISTAIPFP YEYKPGDYRQ TNYLVSKDNQ HYYTANLVIF GPGTNIVENN AVYYKKEDSE N GMGTMSEI WFQPFLTYKY DQFYVDPALE LIKCLIKSLY YLYGIKPSDD LSIPYRLRSE FNSLEYSELD MVDFLISGGT DY KLLNTNP YWFTDNYFIN APKNFEKYKN DYETKIKNNN DIANSIKLYL EQKFKTNVQD IWELNLSYFS TEFEIMMPEI FNN ALNHYH RKEYYVIDYF KNYNINGFIN GQIKTILLLS KYNKNIINKP ELIVNLINEN NSVLMKSNIY GDGLKGTIGN FYAV YKIPY NIGDEYHINS SDSCLDNVDI KEIDNIPPIN DADIYPYRKN CDPFTPVYNI TETKEINTTI PFPVNYLQAQ VTNSN DINL SSDFLKVISS KDRSLVYSFL DNTIDYLDSI KYDGPIDTDK KYYLWLKEIF RNYSFDMTET QEVNTLCGFN KVVPWL GKA LNILNTGNSF IEEFKTLGPI SLINKKENIT MPKIEIDEIP NSMLNLSFKD LSENLFNIFS KNNSYFEKIY YDFLDQW WT QYYSQYFDLI CMAKRSVLAQ ESLIKKIIQK KLSYLIGNSN ISSDNLALMN LTTTNTLRDI SNESQIAMNN VNNFLNNV A ICVFQTNIYP KFISFMEQCI NNINKNTREF IQKCTNITEN EKLQLINQNI FSSLDFDFLN IENLKSLFNS ETGLLIKEE TSPYELVLYA FQEPGNNAIG DASGKNTSIE YSKDIGLVYG INSDALYLNG SNQSISFSND FFENGLTNSF SIYFWLRNLG KDTIKSKLI GSKEDNCGWE IYFQDTGLVF NMIDSNGNEK NIYLSDVSNN SWHYITISVD RLKEQLLIFI DDNLVANESI K EILNIYSS NIISLLSENN PSYIEGLTIL NKPTTSQEVL SNYFKVLNNS YIRDSSEERL EYNKTYQLYN YVFSENPIYE IK QNNNIYL TINNTNNLNL QVSKFKLLSI NPNKQYVQKL DEVIISVLDN MEKYIDISED NRLQLIDNKN NAKKMIISND IFI SNCLTL SYNGKYICLS MKDENHNWMI CNNDMSKYLY LWSFK

UniProtKB: NtnH

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Macromolecule #3: OrfX2

MacromoleculeName: OrfX2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum A (bacteria)
Molecular weightTheoretical: 84.823297 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNNLKPFIYY DWEKTILKNT KENYSINEII PKTFFMELHG TKITNSTLNG TWKSWNLTNE GEGSYPVLKC IIDDGYLDMN FGASSEKIP LKNVWIKLCM KINPNSDGTY SIPEKSSSFY IKDNSLKISK DNLILDKYLN KLMLSYFKNN IKNIEMFINK S RIQTKVVG ...String:
MNNLKPFIYY DWEKTILKNT KENYSINEII PKTFFMELHG TKITNSTLNG TWKSWNLTNE GEGSYPVLKC IIDDGYLDMN FGASSEKIP LKNVWIKLCM KINPNSDGTY SIPEKSSSFY IKDNSLKISK DNLILDKYLN KLMLSYFKNN IKNIEMFINK S RIQTKVVG DLSLLGWNTE NSVSFRTMNE FIKKDNLYPK DFKAVYSYRK MTFTATGTFD SWEMTTGADG RNIRFKCPIK YA VYDLDGD VFNSSTENFL LIQVDLTYFD SKTTINDPTG ENDGKQFNLK IKTNDDKLKN VLIVTYNLTD TDGSMSSEDK DFL SLAFRN WFNENIQQFE QIFAYILLDE TAKIPEYQWL KPTQISYGSA SVETANDEPD LDASIFSAMS MVENNTNSTP SHAV DNRML QLTKTQAAFG ISFPLFIEHF LKQALLSSQF ISVDDIVADI NTLTITNNKQ IIFGKVENSD GKNVDSSLKP GKLKL SLQN NLIVLELFDL TWEQGRGVTG HFDFRQEYEL ALESKSGKQI PILKVHDEPE IEYYVEEAQW KTNEDMIVSA VVGTVF SMI LGASMKLAGS ALSKAGKLIR SKATTIKGRK KIYINRSNVR QLRKDSGATE IELERINRRN SSIAAEDARL ISNNGTT SI QTLGDMKKKP MSTGQRIAIG AKKIAGTAVM FGAVGLGMNF GEMLINYINA MENNDYSAIP GINSFMQQCI GAMQWPDK D SELKVTFGKL QGIYLLGGTL EKNNKTDNK

UniProtKB: OrfX2

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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

Concentration0.5 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
0.2 MMESMES
0.1 MNaClsodium chloride
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 3 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 135181
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9arl:
CryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botulinum strain A1-ST7B, major class

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