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- PDB-4ej5: Crystal structure of the catalytic domain of botulinum neurotoxin... -

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Basic information

Entry
Database: PDB / ID: 4ej5
TitleCrystal structure of the catalytic domain of botulinum neurotoxin BoNT/A wild-type
ComponentsBotulinum neurotoxin A light chain
KeywordsHYDROLASE / Metalloprotease / peptidase M27 superfamily / Clostridial neurotoxin zinc protease / Human target snap-25
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 / extracellular region / zinc ion binding / membrane
Similarity search - Function
Zincin-like / Metalloproteases ("zincins"), catalytic domain like / 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 ...Zincin-like / Metalloproteases ("zincins"), catalytic domain like / 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 / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
CARBONATE ION / IMIDAZOLE / Botulinum neurotoxin type A / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesClostridium botulinum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.87 Å
AuthorsStura, E.A. / Vera, L. / Dive, V.
CitationJournal: J.Biol.Chem. / Year: 2012
Title: Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165.
Authors: Stura, E.A. / Le Roux, L. / Guitot, K. / Garcia, S. / Bregant, S. / Beau, F. / Vera, L. / Collet, G. / Ptchelkine, D. / Bakirci, H. / Dive, V.
History
DepositionApr 6, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 15, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 29, 2012Group: Database references
Revision 1.2Oct 17, 2012Group: Database references
Revision 1.3Dec 21, 2022Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / 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_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_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_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_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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Sep 20, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,4749
Polymers50,9051
Non-polymers5698
Water6,972387
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)49.950, 66.370, 64.720
Angle α, β, γ (deg.)90.00, 98.24, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 50905.379 Da / Num. of mol.: 1 / Fragment: UNP residues 1-425
Source method: isolated from a genetically manipulated source
Details: expression vector between Nde I and Sal I restriction sites, generating a thrombin cleavable N-terminal 6His affinity tag
Source: (gene. exp.) Clostridium botulinum (bacteria) / Strain: Hall ATCC 3502 / Gene: botA, atx, bna / Plasmid: pET28a+ / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta DE3
References: UniProt: P10845, UniProt: P0DPI1*PLUS, bontoxilysin

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Non-polymers , 7 types, 395 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2
#6: Chemical ChemComp-CO3 / CARBONATE ION


Mass: 60.009 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: CO3
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 387 / 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: 41.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6
Details: 8.3% monomethyl polyethylene glycol 2000, 0.17 M lithium sulfate, 6.25% 4,2 methyl pentane diol, 0.1 M imidazole malate, pH 6.0, cryoprotectant: 18% MPEG2K, 22% MPD, 10% DMSO, .050 M bicine, ...Details: 8.3% monomethyl polyethylene glycol 2000, 0.17 M lithium sulfate, 6.25% 4,2 methyl pentane diol, 0.1 M imidazole malate, pH 6.0, cryoprotectant: 18% MPEG2K, 22% MPD, 10% DMSO, .050 M bicine, pH 7.5, VAPOR DIFFUSION, SITTING DROP, temperature 293.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-2 / Wavelength: 0.933 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Sep 29, 2007 / Details: mirrors
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.933 Å / Relative weight: 1
ReflectionResolution: 1.87→42.17 Å / Num. all: 34715 / Num. obs: 34616 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 4.19 % / Biso Wilson estimate: 25.086 Å2 / Rmerge(I) obs: 0.06 / Rsym value: 0.052 / Net I/σ(I): 22.27
Reflection shellResolution: 1.87→1.98 Å / Redundancy: 4.17 % / Rmerge(I) obs: 0.317 / Mean I/σ(I) obs: 4.83 / Num. unique all: 5551 / Rsym value: 0.363 / % possible all: 99.4

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Processing

Software
NameVersionClassification
DNAdata collection
MOLREPphasing
PHENIX(phenix.refine)refinement
XDSdata reduction
XSCALEdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2ILP

2ilp


Resolution: 1.87→42.166 Å / SU ML: 0.48 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 2 / σ(I): -3 / Phase error: 19.62 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2084 1731 5.01 %RANDOM
Rwork0.1611 ---
obs0.1634 34577 99.67 %-
all-34577 --
Solvent computationShrinkage radii: 0.72 Å / VDW probe radii: 1 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 62.788 Å2 / ksol: 0.4 e/Å3
Displacement parametersBiso mean: 24.268 Å2
Baniso -1Baniso -2Baniso -3
1-1.8527 Å2-0 Å20.133 Å2
2---0.7657 Å20 Å2
3----1.087 Å2
Refinement stepCycle: LAST / Resolution: 1.87→42.166 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3501 0 33 387 3921
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073663
X-RAY DIFFRACTIONf_angle_d1.0414957
X-RAY DIFFRACTIONf_dihedral_angle_d18.1341327
X-RAY DIFFRACTIONf_chiral_restr0.075532
X-RAY DIFFRACTIONf_plane_restr0.005647
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.87-1.9260.28751430.24072727X-RAY DIFFRACTION99
1.926-1.98820.25221440.20442735X-RAY DIFFRACTION100
1.9882-2.05920.24121430.18032719X-RAY DIFFRACTION100
2.0592-2.14170.24751440.17272732X-RAY DIFFRACTION100
2.1417-2.23910.23551450.16592751X-RAY DIFFRACTION100
2.2391-2.35720.24851420.16132703X-RAY DIFFRACTION100
2.3572-2.50490.21631450.16242750X-RAY DIFFRACTION100
2.5049-2.69820.19281430.15722710X-RAY DIFFRACTION100
2.6982-2.96970.20631450.15232756X-RAY DIFFRACTION100
2.9697-3.39930.18981440.15022749X-RAY DIFFRACTION100
3.3993-4.28210.17611450.13562757X-RAY DIFFRACTION100
4.2821-42.1660.18771480.1642757X-RAY DIFFRACTION98

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