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- PDB-2w2d: Crystal Structure of a Catalytically Active, Non-toxic Endopeptid... -

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Basic information

Entry
Database: PDB / ID: 2w2d
TitleCrystal Structure of a Catalytically Active, Non-toxic Endopeptidase Derivative of Clostridium botulinum Toxin A
Components(BOTULINUM NEUROTOXIN A ...) x 2
KeywordsHYDROLASE / METALLOPROTEASE / MEMBRANE DOMAIN / PROTEIN ENGINEERING / NEUROTOXIN / METAL-BINDING / TRANSMEMBRANE / PHARMACEUTICAL / ZINC PROTEASE / MIXED ALPHA AND BETA / BONT / MEMBRANE / SECRETED / PROTEASE
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 / metalloendopeptidase activity / toxin activity / membrane => GO:0016020 ...host cell junction / negative regulation of neurotransmitter secretion / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / membrane => GO:0016020 / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / membrane
Similarity search - Function
Clostridium botulinum neurotoxin B, "coiled-coil" domain / Clostridium botulinum neurotoxin b, "coiled-coil" domain / 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 N-terminal ...Clostridium botulinum neurotoxin B, "coiled-coil" domain / Clostridium botulinum neurotoxin b, "coiled-coil" domain / 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 N-terminal / Clostridium neurotoxin, receptor-binding C-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 / Up-down Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / Botulinum neurotoxin type A / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesCLOSTRIDIUM BOTULINUM (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsMasuyer, G. / Thiyagarajan, N. / James, P.L. / Marks, P.M.H. / Chaddock, J.A. / Acharya, K.R.
CitationJournal: Biochem.Biophys.Res.Commun. / Year: 2009
Title: Crystal Structure of a Catalytically Active, Non-Toxic Endopeptidase Derivative of Clostridium Botulinum Toxin A.
Authors: Masuyer, G. / Thiyagarajan, N. / James, P.L. / Marks, P.M.H. / Chaddock, J.A. / Acharya, K.R.
History
DepositionOct 29, 2008Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 24, 2009Provider: repository / Type: Initial release
Revision 1.1Mar 28, 2012Group: Database references / Derived calculations ...Database references / Derived calculations / Non-polymer description / Other / Refinement description / Structure summary / Version format compliance
Revision 1.2May 4, 2022Group: Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _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_conn.ptnr2_label_seq_id / _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: BOTULINUM NEUROTOXIN A HEAVY CHAIN
C: BOTULINUM NEUROTOXIN A LIGHT CHAIN
D: BOTULINUM NEUROTOXIN A HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,25119
Polymers202,1104
Non-polymers1,14115
Water5,549308
1
A: BOTULINUM NEUROTOXIN A LIGHT CHAIN
B: BOTULINUM NEUROTOXIN A HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,84412
Polymers101,0552
Non-polymers78910
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11890 Å2
ΔGint-128 kcal/mol
Surface area36480 Å2
MethodPISA
2
C: BOTULINUM NEUROTOXIN A LIGHT CHAIN
D: BOTULINUM NEUROTOXIN A HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,4077
Polymers101,0552
Non-polymers3525
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11160 Å2
ΔGint-121.7 kcal/mol
Surface area36730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.365, 156.909, 211.651
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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BOTULINUM NEUROTOXIN A ... , 2 types, 4 molecules ACBD

#1: Protein BOTULINUM NEUROTOXIN A LIGHT CHAIN / BONTOXILYSIN-A


Mass: 51510.227 Da / Num. of mol.: 2 / Fragment: RESIDUES 1-442
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM BOTULINUM (bacteria) / Plasmid: MODIFIED PMAL-C2X / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21
References: UniProt: A5HZZ9, UniProt: P0DPI1*PLUS, bontoxilysin
#2: Protein BOTULINUM NEUROTOXIN A HEAVY CHAIN / BONTOXILYSIN-A


Mass: 49544.824 Da / Num. of mol.: 2 / Fragment: RESIDUES 447-877
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM BOTULINUM (bacteria) / Plasmid: MODIFIED PMAL-C2X / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21
References: UniProt: A5HZZ9, UniProt: P0DPI1*PLUS, bontoxilysin

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Non-polymers , 6 types, 323 molecules

#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#7: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 308 / 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: 3.25 Å3/Da / Density % sol: 62.2 % / Description: NONE
Crystal growpH: 8.5
Details: PROTEIN: 0.05 M HEPES PH 7.2 AND 0.2 M NACL MOTHER LIQUOR: 15% SUCROSE, 0.1 M TRIS PH 8.5, 1.5 M AMMONIUM SULPHATE.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.826, 1.55
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 9, 2008 / Details: MIRRORS
RadiationMonochromator: NI FILTER / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.8261
21.551
ReflectionResolution: 2.59→50 Å / Num. obs: 82260 / % possible obs: 97.1 % / Observed criterion σ(I): 0 / Redundancy: 1.9 % / Biso Wilson estimate: 36.8 Å2 / Rmerge(I) obs: 0.11 / Net I/σ(I): 11.5
Reflection shellResolution: 2.59→2.68 Å / Redundancy: 1.8 % / Rmerge(I) obs: 0.55 / Mean I/σ(I) obs: 2 / % possible all: 98.4

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Processing

Software
NameVersionClassification
CNS1.2refinement
HKL-2000data reduction
HKL-2000data scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.59→49.11 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 50365.68 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
Details: BULK SOLVENT MODEL USED SOME OF THE DISORDERED REGIONS WERE MODELLED STEREOCHEMICALLY AND AT SOME PLACES THE SIDECHAINS WERE REMOVED KEEPING THE MAIN CHAIN
RfactorNum. reflection% reflectionSelection details
Rfree0.253 1116 1.5 %RANDOM
Rwork0.212 ---
obs0.212 74760 91.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 51.2055 Å2 / ksol: 0.4 e/Å3
Displacement parametersBiso mean: 41 Å2
Baniso -1Baniso -2Baniso -3
1--2.89 Å20 Å20 Å2
2--3.7 Å20 Å2
3----0.81 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.37 Å0.31 Å
Luzzati d res low-5 Å
Luzzati sigma a0.38 Å0.34 Å
Refinement stepCycle: LAST / Resolution: 2.59→49.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13793 0 59 308 14160
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d21.8
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.77
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
Refine LS restraints NCSNCS model details: NONE
LS refinement shellResolution: 2.59→2.75 Å / Rfactor Rfree error: 0.025 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.329 178 1.6 %
Rwork0.283 11191 -
obs--84.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMGOL-ACT.TOP
X-RAY DIFFRACTION2ION.PARAM
X-RAY DIFFRACTION3WATER_REP.PARAM
X-RAY DIFFRACTION4GOL-ACT.PARAM

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