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

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Basic information

Entry
Database: PDB / ID: 4kuf
TitleCrystal structure of the catalytic domain of botulinum neurotoxin BoNT/A C134 mutant with MTSEA modified Cys-165 causing stretch disorder
ComponentsBotulinum neurotoxin A light chain
KeywordsHYDROLASE / Clostridial neurotoxin zinc protease / Peptidase_M27 / SNAP 25 / covalent inhibition
Function / homology
Function and homology information


host cell junction / bontoxilysin / negative regulation of neurotransmitter secretion / 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 / bontoxilysin / negative regulation of neurotransmitter secretion / 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
(2S)-2-hydroxybutanedioic acid / Botulinum neurotoxin type A / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesClostridium botulinum A (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / RIGID BODY / Resolution: 1.697 Å
AuthorsStura, E.A. / Vera, L. / Ptchelkine, D. / Bakirci, H. / Garcia, S. / 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 22, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 9, 2014Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Data collection / Derived calculations / Refinement description
Category: 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_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

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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)52,17319
Polymers50,9641
Non-polymers1,20918
Water5,981332
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)65.460, 65.460, 201.620
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-925-

HOH

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 50964.449 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

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Non-polymers , 7 types, 350 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical
ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#4: Chemical ChemComp-LMR / (2S)-2-hydroxybutanedioic acid / L-Malate


Mass: 134.087 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6O5
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 332 / 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: 2.12 Å3/Da / Density % sol: 41.95 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: LCA-C134S-MTSEA at 3 mg/ml in 0.010 M Tris-HCl, pH 7.9, 0.050 M Li2SO4, DROPS - 3+3 micro-l, RESERVOIR - 42% MPEG-550, 0.1 M Li2SO4, 0.2 M imidazole malate, pH 5.5, CRYOSOLUTION - 18% ...Details: LCA-C134S-MTSEA at 3 mg/ml in 0.010 M Tris-HCl, pH 7.9, 0.050 M Li2SO4, DROPS - 3+3 micro-l, RESERVOIR - 42% MPEG-550, 0.1 M Li2SO4, 0.2 M imidazole malate, pH 5.5, CRYOSOLUTION - 18% MPEG2K, 22% MPD, 10% DMSO, 0.050 M bicine, pH 7.5, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-4 / Wavelength: 0.9395 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 30, 2009 / Details: mirrors
RadiationMonochromator: SAGITALLY FOCUSED Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9395 Å / Relative weight: 1
ReflectionResolution: 1.7→50 Å / Num. all: 49624 / Num. obs: 49488 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 15.6 % / Biso Wilson estimate: 29.91 Å2 / Rmerge(I) obs: 0.147 / Rsym value: 0.143 / Net I/σ(I): 16.17
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allRsym value% possible all
1.7-1.815.61.8861.5778421.82498.5
1.8-1.9216.11.1092.9574231.073100
1.92-2.0816.10.6125.6669120.593100
2.08-2.28160.34310.363990.332100
2.28-2.5415.80.22315.3258190.216100
2.54-2.9315.50.13822.9451820.134100
2.93-3.5914.80.07835.9544340.076100
3.59-5.0614.30.05151.3835150.049100
5.06-5014.20.03854.520980.03699.2

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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: RIGID BODY
Starting model: 4ELC

4elc


Resolution: 1.697→39.937 Å / SU ML: 0.21 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 2 / σ(I): -3 / Phase error: 20.85 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2309 2475 5 %RANDOM
Rwork0.1802 ---
obs0.1827 49485 99.73 %-
all-49488 --
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.697→39.937 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3458 0 70 332 3860
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0083693
X-RAY DIFFRACTIONf_angle_d1.1264993
X-RAY DIFFRACTIONf_dihedral_angle_d14.8861376
X-RAY DIFFRACTIONf_chiral_restr0.079536
X-RAY DIFFRACTIONf_plane_restr0.005649
LS refinement shell

Refine-ID: X-RAY DIFFRACTION

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection obs% reflection obs (%)
1.6967-1.72930.33331290.31992452258196
1.7293-1.76460.30321360.27725742710100
1.7646-1.8030.34451340.27325422676100
1.803-1.84490.28061350.236925702705100
1.8449-1.8910.28631350.222525732708100
1.891-1.94220.2911360.213725892711100
1.9422-1.99930.2541360.204925772713100
1.9993-2.06380.24721360.185125812717100
2.0638-2.13760.22941360.181425822731100
2.1376-2.22320.22831360.175225952718100
2.2232-2.32440.23081390.178926242763100
2.3244-2.44690.22691370.172326052763100
2.4469-2.60020.21841370.177126092742100
2.6002-2.80090.25241380.175926322770100
2.8009-3.08260.24271400.170926472787100
3.0826-3.52850.2391400.161826602800100
3.5285-4.44460.1791430.142827252868100
4.4446-39.94820.20081520.178128733025100

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