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- PDB-8qft: X-ray structure of non-toxic non-hemagglutinin (NTNH) protein fro... -

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Basic information

Entry
Database: PDB / ID: 8qft
TitleX-ray structure of non-toxic non-hemagglutinin (NTNH) protein from botulinum neurotoxin serotype X
Componentsnon-toxic non-hemagglutinin protein X
KeywordsTOXIN / botulinum neurotoxin / progenitor toxin complex / Clostridium botulinum / strain 111
Biological speciesClostridium botulinum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsSkerlova, J. / Masuyer, G. / Stenmark, P.
Funding support Sweden, European Union, 3items
OrganizationGrant numberCountry
Swedish Research Council2018-03406 Sweden
Swedish Research Council2022-03681 Sweden
European Regional Development FundCZ.02.2.69/0.0/0.0/16_027/0008477European Union
CitationJournal: Commun Chem / Year: 2024
Title: Activity of botulinum neurotoxin X and its structure when shielded by a non-toxic non-hemagglutinin protein.
Authors: Markel Martínez-Carranza / Jana Škerlová / Pyung-Gang Lee / Jie Zhang / Ajda Krč / Abhishek Sirohiwal / Dave Burgin / Mark Elliott / Jules Philippe / Sarah Donald / Fraser Hornby / Linda ...Authors: Markel Martínez-Carranza / Jana Škerlová / Pyung-Gang Lee / Jie Zhang / Ajda Krč / Abhishek Sirohiwal / Dave Burgin / Mark Elliott / Jules Philippe / Sarah Donald / Fraser Hornby / Linda Henriksson / Geoffrey Masuyer / Ville R I Kaila / Matthew Beard / Min Dong / Pål Stenmark /
Abstract: Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein ...Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.
History
DepositionSep 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: 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.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: non-toxic non-hemagglutinin protein X


Theoretical massNumber of molelcules
Total (without water)137,3951
Polymers137,3951
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area51870 Å2
Unit cell
Length a, b, c (Å)174.106, 174.106, 138.023
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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Components

#1: Protein non-toxic non-hemagglutinin protein X


Mass: 137394.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum (bacteria) / Strain: 111 / Production host: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.4 Å3/Da / Density % sol: 72.02 % / Description: clusters of hexagonal rods
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 15 mg/ml NTNH/X in 20 mM MES pH 5.5, 150 mM NaCl, 0.5 mM TCEP mixed in 2:1 volume ratio with 90 mM MES/imidazole pH 6.5, 9% (w/v) PEG 4,000, 18% (v/v) glycerol, and 3% (w/v) dextran sulfate sodium salt

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jan 27, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.3→87.05 Å / Num. obs: 36727 / % possible obs: 100 % / Redundancy: 19.3 % / CC1/2: 0.999 / Rmerge(I) obs: 0.202 / Rpim(I) all: 0.047 / Rrim(I) all: 0.208 / Χ2: 1.05 / Net I/σ(I): 7 / Num. measured all: 709704
Reflection shellResolution: 3.3→3.45 Å / % possible obs: 100 % / Redundancy: 16.5 % / Rmerge(I) obs: 2.772 / Num. measured all: 72911 / Num. unique obs: 4424 / CC1/2: 0.924 / Rpim(I) all: 0.699 / Rrim(I) all: 2.859 / Χ2: 1.13 / Net I/σ(I) obs: 0.6

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0405refinement
Aimlessdata scaling
DIALSdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.3→73.74 Å / Cor.coef. Fo:Fc: 0.938 / Cor.coef. Fo:Fc free: 0.906 / Cross valid method: FREE R-VALUE / ESU R Free: 0.593
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.3326 1939 5.308 %
Rwork0.2763 34592 -
all0.279 --
obs-36531 99.526 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 220.051 Å2
Baniso -1Baniso -2Baniso -3
1-9.301 Å24.651 Å2-0 Å2
2--9.301 Å20 Å2
3----30.173 Å2
Refinement stepCycle: LAST / Resolution: 3.3→73.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9710 0 0 0 9710
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.0129925
X-RAY DIFFRACTIONr_bond_other_d0.0010.0169263
X-RAY DIFFRACTIONr_angle_refined_deg1.0441.63613473
X-RAY DIFFRACTIONr_angle_other_deg0.6161.56421333
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.16751173
X-RAY DIFFRACTIONr_dihedral_angle_2_deg3.892519
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.157101763
X-RAY DIFFRACTIONr_dihedral_angle_6_deg14.16810531
X-RAY DIFFRACTIONr_chiral_restr0.0490.21494
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.0211487
X-RAY DIFFRACTIONr_gen_planes_other0.0050.022365
X-RAY DIFFRACTIONr_nbd_refined0.1960.22402
X-RAY DIFFRACTIONr_symmetry_nbd_other0.230.29849
X-RAY DIFFRACTIONr_nbtor_refined0.1860.25063
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.080.25120
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1910.2253
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.090.28
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.2660.228
X-RAY DIFFRACTIONr_nbd_other0.2830.278
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.2230.27
X-RAY DIFFRACTIONr_mcbond_it17.50621.7574695
X-RAY DIFFRACTIONr_mcbond_other17.50621.7574695
X-RAY DIFFRACTIONr_mcangle_it26.68639.1065867
X-RAY DIFFRACTIONr_mcangle_other26.68439.1045868
X-RAY DIFFRACTIONr_scbond_it16.78322.3955230
X-RAY DIFFRACTIONr_scbond_other16.78222.3965231
X-RAY DIFFRACTIONr_scangle_it26.53641.0037606
X-RAY DIFFRACTIONr_scangle_other26.53541.0027607
X-RAY DIFFRACTIONr_lrange_it37.621263.96941330
X-RAY DIFFRACTIONr_lrange_other37.622263.96741331
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
3.3-3.3860.5571190.5525410.55126780.8210.82199.32790.551
3.386-3.4780.5341630.51724280.51826100.8160.83899.2720.505
3.478-3.5790.5541290.45823820.46225310.8470.84599.20980.437
3.579-3.6890.4281370.4323160.4324700.8540.85299.31170.416
3.689-3.8090.4211360.38922530.39124080.8540.86999.2110.369
3.809-3.9430.4461570.36921640.37423360.8520.88799.35790.346
3.943-4.0910.3761340.31320660.31622140.8920.91399.36770.289
4.091-4.2570.3381130.2420730.24421930.9260.9599.68080.221
4.257-4.4460.276880.22219650.22420610.9350.96299.61180.205
4.446-4.6620.316790.23218870.23619780.9390.96499.39330.216
4.662-4.9130.249870.2117980.21218890.9620.97399.78820.198
4.913-5.210.283960.2316840.23317860.9460.96699.66410.216
5.21-5.5680.361830.26716130.27116970.920.9699.94110.251
5.568-6.0110.325840.27115100.27415980.9420.9699.74970.26
6.011-6.580.315870.26313580.26614460.9380.9699.93080.254
6.58-7.350.382750.26612470.27313220.9240.9591000.264
7.35-8.4730.266570.21911230.22211800.9530.971000.221
8.473-10.3430.213550.1859520.18610070.9760.9811000.19
10.343-14.4850.216350.2017720.2028070.9740.9771000.208
14.485-73.740.44250.3474600.3514890.810.91899.1820.522

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