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- PDB-1e1h: Crystal Structure of recombinant Botulinum Neurotoxin Type A Ligh... -

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Basic information

Entry
Database: PDB / ID: 1e1h
TitleCrystal Structure of recombinant Botulinum Neurotoxin Type A Light Chain, self-inhibiting Zn endopeptidase.
Components(BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN) x 2
KeywordsHYDROLASE / NEUROTOXIN / ZN-ENDOPEPTIDASE / COMPLEX / SUBSTRATE BOUND / BOTULINUM / INHIBITOR BOUND
Function / homology
Function and homology information


bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region ...bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region / zinc ion binding / membrane
Similarity search - Function
Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #540 / Metalloproteases ("zincins"), catalytic domain fold / Clostridium neurotoxins / 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 ...Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #540 / Metalloproteases ("zincins"), catalytic domain fold / Clostridium neurotoxins / 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 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / Few Secondary Structures / Irregular / Concanavalin A-like lectin/glucanase domain superfamily / Alpha-Beta Complex / Up-down Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Botulinum neurotoxin type A2
Similarity search - Component
Biological speciesCLOSTRIDIUM BOTULINUM (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsKnapp, M. / Rupp, B.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2004
Title: Crystal Structure of Clostridium Botulinum Neurotoxin Protease in a Product-Bound State: Evidence for Noncanonical Zinc Protease Activity
Authors: Segelke, B.W. / Knapp, M. / Kadhkodayan, S. / Balhorn, R. / Rupp, B.
History
DepositionMay 8, 2000Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2003Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Apr 10, 2019Group: Data collection / Other / Source and taxonomy
Category: entity_src_gen / pdbx_database_proc / pdbx_database_status
Item: _entity_src_gen.pdbx_host_org_cell_line / _entity_src_gen.pdbx_host_org_variant / _pdbx_database_status.recvd_author_approval
Revision 1.4May 8, 2019Group: Data collection / Experimental preparation
Category: database_PDB_rev / database_PDB_rev_record / exptl_crystal_grow
Item: _exptl_crystal_grow.method
Revision 1.5Dec 6, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / 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 / _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_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 TYPE A LIGHT CHAIN
B: BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN
C: BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN
D: BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,5576
Polymers105,4264
Non-polymers1312
Water13,836768
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18930 Å2
ΔGint-197.3 kcal/mol
Surface area34360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.064, 94.263, 100.156
Angle α, β, γ (deg.)90.00, 103.52, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(-0.8397, 0.5389, -0.0674), (0.5406, 0.8178, -0.1973), (-0.0512, -0.2021, -0.978)3.2947, 3.9508, 48.0381
2given(-0.8397, 0.5406, -0.0512), (0.5389, 0.8178, -0.2021), (-0.0674, -0.1973, -0.978)-3.2947, -3.9508, -48.0381

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Components

#1: Protein BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN / BONT/A LC


Mass: 32153.004 Da / Num. of mol.: 2 / Fragment: RESIDUES 10-250
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM BOTULINUM (bacteria) / Strain: KYOTO-F / Production host: ESCHERICHIA COLI (E. coli) / Variant (production host): BLR DE-3 / References: UniProt: Q45894, bontoxilysin
#2: Protein BOTULINUM NEUROTOXIN TYPE A LIGHT CHAIN / BONT/A LC


Mass: 20560.143 Da / Num. of mol.: 2 / Fragment: RESIDUES 252-416 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: HOMODIMER, CONTAINING CLEAVED SUBSTRATE ANALOG (LOOP 245-255) IN ACTIVE SITE
Source: (gene. exp.) CLOSTRIDIUM BOTULINUM (bacteria) / Strain: KYOTO-F / Production host: ESCHERICHIA COLI (E. coli) / Variant (production host): BLR DE-3 / References: UniProt: Q45894, bontoxilysin
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 768 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTER TYR: PEPTIDE CHAIN CLEAVED AT AA 249 AND 250. HIS: PEPTIDE CHAIN CLEAVED AT AA 249 AND 250.
Sequence detailsEXPERIMENTAL PROTEIN HAS 6XHIS-TAG AND S-TAG AT N-TERMINUS FOLLOWED BY RESIDUES 9-415 OF NCBI: ...EXPERIMENTAL PROTEIN HAS 6XHIS-TAG AND S-TAG AT N-TERMINUS FOLLOWED BY RESIDUES 9-415 OF NCBI:CBNTOXA, THEN A C-TERM 6XHIS-TAG WITH A 2 RESIDUE LEADER (LE). SEQUENCE IN COORDINATE FILE IS NUMBERED AS IN NCBI:CBNTOXA RESIDUES 7&8 ARE FROM THE AFFINITY TAG.

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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: 2.91 Å3/Da / Density % sol: 53 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 4.6
Details: HANGING DROP VAPOUR DIFFUSION, DROP: 4UL 5MG/ML PROTEIN & 2UL WELL. PROTEIN: 0.05M TRIS PH 8.0,10% GLYCEROL, 0.1% TRITON X-100,1.0MM 2-ME,4% XYLITOL. WELL: 0.2M (NH4)2SO4,0.1M NAOAC PH 4.6, 25% PEG4000.
Crystal grow
*PLUS
Temperature: 22 ℃ / pH: 8 / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
15 mg/mlprotein1drop
20.1 %triton X-1001drop
325 mMTris1droppH8.0
410 %(v/v)glycerol1drop
51 mMMPD1drop
60.2 Mammonium sulfate1reservoir
70.1 Msodium acetate1reservoirpH4.6
825 %(w/v)PEG40001reservoir
93 %xylitol1reservoir

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Data collection

DiffractionMean temperature: 125 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.1
DetectorType: ADSC CCD / Detector: CCD / Date: Jan 15, 2000 / Details: DOUBLE FOCUSSING
RadiationMonochromator: SI / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.8→19.34 Å / Num. obs: 93168 / % possible obs: 95.2 % / Observed criterion σ(I): 0 / Redundancy: 1.9 % / Biso Wilson estimate: 24.47 Å2 / Rsym value: 0.042 / Net I/σ(I): 7.9
Reflection shellResolution: 1.8→1.9 Å / Redundancy: 1.9 % / Mean I/σ(I) obs: 1.7 / Rsym value: 0.383 / % possible all: 95.2
Reflection
*PLUS
Highest resolution: 1.8 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.042
Reflection shell
*PLUS
Highest resolution: 1.8 Å / Lowest resolution: 1.9 Å / Redundancy: 1.9 % / Num. unique obs: 6918 / Rmerge(I) obs: 0.383 / Mean I/σ(I) obs: 1.7

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Processing

Software
NameClassification
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling
EPMRphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: LC COORDINATES OF PDB ENTRY 3BTA

3bta


Resolution: 1.8→19.34 Å / SU B: 2.746 / SU ML: 0.085 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.129 / ESU R Free: 0.127
RfactorNum. reflection% reflectionSelection details
Rfree0.237 4649 5 %RANDOM
Rwork0.196 ---
obs-92076 95.2 %-
Displacement parametersBiso mean: 31.5 Å2
Refinement stepCycle: LAST / Resolution: 1.8→19.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6460 0 2 768 7230
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.020.02
X-RAY DIFFRACTIONp_angle_d0.0190.02
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d0.0270.03
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it1.9042
X-RAY DIFFRACTIONp_mcangle_it2.6463
X-RAY DIFFRACTIONp_scbond_it3.4543
X-RAY DIFFRACTIONp_scangle_it5.0514
X-RAY DIFFRACTIONp_plane_restr0.0180.03
X-RAY DIFFRACTIONp_chiral_restr0.1910.15
X-RAY DIFFRACTIONp_singtor_nbd0.1820.3
X-RAY DIFFRACTIONp_multtor_nbd0.2760.3
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd0.1430.3
X-RAY DIFFRACTIONp_planar_tor5.217
X-RAY DIFFRACTIONp_staggered_tor16.9515
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor26.2120
X-RAY DIFFRACTIONp_special_tor
Refinement
*PLUS
Highest resolution: 1.8 Å
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_bond_d0.02
X-RAY DIFFRACTIONp_angle_d
X-RAY DIFFRACTIONp_angle_deg1.85

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