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- PDB-4ktx: Crystal structure of the catalytic domain of botulinum neurotoxin... -

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Basic information

Entry
Database: PDB / ID: 4ktx
TitleCrystal structure of the catalytic domain of botulinum neurotoxin BoNT/A C134S mutant with covalent inhibitor that modifies Cys-165 causing disorder in 167-174 stretch
Components
  • Botulinum neurotoxin A light chain
  • Peptide inhibitor MPT-DPP-ARG-G-LEU-NH2
KeywordsHYDROLASE/HYDROLASE INHIBITOR / Clostridial neurotoxin zinc protease / Peptidase_M27 / SNAP 25 / covalent inhibition / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


host cell junction / negative regulation of neurotransmitter secretion / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / membrane => GO:0016020 / metalloendopeptidase activity / toxin activity ...host cell junction / negative regulation of neurotransmitter secretion / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / membrane => GO:0016020 / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / membrane
Similarity search - Function
Zincin-like / Metalloproteases ("zincins"), catalytic domain like / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, C-terminal receptor binding ...Zincin-like / Metalloproteases ("zincins"), catalytic domain like / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, C-terminal receptor binding / 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 / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Peptide inhibitor MPT-DPP-ARG-G-LEU-NH2 / S-1,2-PROPANEDIOL / Botulinum neurotoxin type A / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesClostridium botulinum A (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsStura, E.A. / Vera, L. / Guitot, K. / Dive, V.
CitationJournal: To be Published
Title: Covalent modification of the active site cysteine stresses Clostridium botulinum neurotoxin A
Authors: Guitot, K. / Vera, L. / Le Roux, L. / Bregant, S. / Ptchelkine, D. / Beau, F. / Stura, E.A. / Dive, V.
History
DepositionMay 21, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 15, 2014Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Refinement description / Source and taxonomy
Category: pdbx_entity_src_syn / pdbx_validate_polymer_linkage ...pdbx_entity_src_syn / pdbx_validate_polymer_linkage / software / struct_conn
Item: _software.classification / _software.name ..._software.classification / _software.name / _software.version / _struct_conn.pdbx_leaving_atom_flag
Revision 1.2Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Botulinum neurotoxin A light chain
B: Peptide inhibitor MPT-DPP-ARG-G-LEU-NH2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,9218
Polymers51,4072
Non-polymers5146
Water2,414134
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2360 Å2
ΔGint-54 kcal/mol
Surface area19420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.410, 65.410, 200.970
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-706-

HOH

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Components

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Protein / Protein/peptide , 2 types, 2 molecules AB

#1: Protein Botulinum neurotoxin A light chain / Botulinum neurotoxin type A / BoNT/A / Bontoxilysin-A / BOTOX


Mass: 50889.316 Da / Num. of mol.: 1 / Fragment: Catalytic domain residues 1-425 / Mutation: C134S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A (bacteria) / Strain: strain Hall / ATCC 3502 / NCTC 13319 / Type A / Gene: botA, CBO0806, CLC_0862 / Plasmid: pET28a+ / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta De3
References: UniProt: A5HZZ9, UniProt: P0DPI1*PLUS, bontoxilysin
#2: Protein/peptide Peptide inhibitor MPT-DPP-ARG-G-LEU-NH2


Type: Peptide-like / Class: Inhibitor / Mass: 517.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: Peptide inhibitor MPT-DPP-ARG-G-LEU-NH2

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Non-polymers , 5 types, 140 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-PGO / S-1,2-PROPANEDIOL


Mass: 76.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O2
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 134 / Source method: isolated from a natural source / Formula: H2O

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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: 2.09 Å3/Da / Density % sol: 41.17 %
Crystal growTemperature: 293 K / pH: 6
Details: Protein - preformed complex LCA_C134S-KG13 at 10 mg/mL, Reservoir - 4% PEG 4000, 0.1 M imidazole malate, Cryoprotectant 40% CryoProtX CM3, 18% MPEG 2K, 0.1 M PCTP (Na propionate, Na ...Details: Protein - preformed complex LCA_C134S-KG13 at 10 mg/mL, Reservoir - 4% PEG 4000, 0.1 M imidazole malate, Cryoprotectant 40% CryoProtX CM3, 18% MPEG 2K, 0.1 M PCTP (Na propionate, Na cacodylate, Bis-Tris-propane) (50% pH 4 / 50% pH 9.5), VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.9724
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jun 13, 2011 / Details: MIRRORS
RadiationMonochromator: HORIZONTALLY DIFFRACTING SI (111) MONOCHROMATOR AND PT COATED MIRRORS IN KIRKPATRICK-BAEZ GEOMETRY FOR FOCUSING
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9724 Å / Relative weight: 1
ReflectionResolution: 2.59→50 Å / Num. obs: 14292 / % possible obs: 99.2 % / Observed criterion σ(I): -3 / Redundancy: 12.76 % / Biso Wilson estimate: 53.11 Å2 / Rmerge(I) obs: 0.446 / Rsym value: 0.439 / Net I/σ(I): 9.03
Reflection shellResolution: 2.59→2.74 Å / Redundancy: 12.4 % / Rmerge(I) obs: 2.479 / Mean I/σ(I) obs: 1.34 / Rsym value: 2.377 / % possible all: 95.5

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Processing

Software
NameVersionClassification
PHENIX(phenix.refine: 1.8_1069)refinement
REFMACrefinement
DNAdata collection
XDSdata reduction
XDSdata scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4ELC

4elc


Resolution: 2.59→46.8 Å / SU ML: 0.46 / Isotropic thermal model: Isotropic / σ(F): 2.01 / Phase error: 28.51 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.298 715 5 %
Rwork0.202 --
obs0.207 14291 99.3 %
all-14292 -
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.59→46.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3499 0 29 134 3662
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013628
X-RAY DIFFRACTIONf_angle_d1.4584900
X-RAY DIFFRACTIONf_dihedral_angle_d16.1371336
X-RAY DIFFRACTIONf_chiral_restr0.057528
X-RAY DIFFRACTIONf_plane_restr0.008634
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.59-2.78840.37951350.25372566X-RAY DIFFRACTION96
2.7884-3.0690.34181410.26912672X-RAY DIFFRACTION100
3.069-3.51290.32471410.21712692X-RAY DIFFRACTION100
3.5129-4.42540.25091450.16822741X-RAY DIFFRACTION100
4.4254-46.80810.28691530.18532905X-RAY DIFFRACTION100

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