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- PDB-6g5k: Crystal structure of the binding domain of Botulinum Neurotoxin t... -

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Basic information

Entry
Database: PDB / ID: 6g5k
TitleCrystal structure of the binding domain of Botulinum Neurotoxin type B in complex with human synaptotagmin 1
Components
  • Botulinum neurotoxin type B
  • Synaptotagmin-1
KeywordsTOXIN / botulinum toxin / neurotoxin / protein engineering / receptor binding
Function / homology
Function and homology information


clathrin-sculpted acetylcholine transport vesicle membrane / clathrin-sculpted glutamate transport vesicle membrane / Toxicity of botulinum toxin type G (botG) / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / clathrin-sculpted monoamine transport vesicle membrane / calcium-dependent activation of synaptic vesicle fusion / regulation of regulated secretory pathway ...clathrin-sculpted acetylcholine transport vesicle membrane / clathrin-sculpted glutamate transport vesicle membrane / Toxicity of botulinum toxin type G (botG) / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / clathrin-sculpted monoamine transport vesicle membrane / calcium-dependent activation of synaptic vesicle fusion / regulation of regulated secretory pathway / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / calcium ion sensor activity / Toxicity of botulinum toxin type B (botB) / spontaneous neurotransmitter secretion / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane / dense core granule / chromaffin granule membrane / GABA synthesis, release, reuptake and degradation / Dopamine Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / calcium ion-regulated exocytosis of neurotransmitter / vesicle docking / regulation of calcium ion-dependent exocytosis / exocytic vesicle / positive regulation of dopamine secretion / protein heterooligomerization / Glutamate Neurotransmitter Release Cycle / bontoxilysin / host cell presynaptic membrane / neurotransmitter secretion / regulation of exocytosis / positive regulation of dendrite extension / neuron projection terminus / calcium-dependent phospholipid binding / host cell cytoplasmic vesicle / Neurexins and neuroligins / syntaxin-1 binding / low-density lipoprotein particle receptor binding / syntaxin binding / host cell cytosol / clathrin binding / phosphatidylserine binding / regulation of dopamine secretion / excitatory synapse / synaptic vesicle endocytosis / protein transmembrane transporter activity / detection of calcium ion / positive regulation of synaptic transmission / regulation of synaptic transmission, glutamatergic / phosphatidylinositol-4,5-bisphosphate binding / hippocampal mossy fiber to CA3 synapse / cellular response to calcium ion / SNARE binding / clathrin-coated endocytic vesicle membrane / metalloendopeptidase activity / synaptic vesicle membrane / calcium-dependent protein binding / synaptic vesicle / Cargo recognition for clathrin-mediated endocytosis / presynaptic membrane / Clathrin-mediated endocytosis / toxin activity / chemical synaptic transmission / cell differentiation / calmodulin binding / neuron projection / protein heterodimerization activity / axon / lipid binding / glutamatergic synapse / calcium ion binding / host cell plasma membrane / Golgi apparatus / proteolysis / zinc ion binding / extracellular region / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Synaptotagmin / 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 ...Synaptotagmin / 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 / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / Trefoil (Acidic Fibroblast Growth Factor, subunit A) - #50 / Trefoil (Acidic Fibroblast Growth Factor, subunit A) / Trefoil / C2 domain superfamily / Jelly Rolls - #200 / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Botulinum neurotoxin type B / Synaptotagmin-1
Similarity search - Component
Biological speciesClostridium botulinum (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsMasuyer, G. / Elliot, M. / Favre-Guilmard, C. / Liu, S.M. / Maignel, J. / Beard, M. / Carre, D. / Kalinichev, M. / Lezmi, S. / Mir, I. ...Masuyer, G. / Elliot, M. / Favre-Guilmard, C. / Liu, S.M. / Maignel, J. / Beard, M. / Carre, D. / Kalinichev, M. / Lezmi, S. / Mir, I. / Nicoleau, C. / Palan, S. / Perier, C. / Raban, E. / Dong, M. / Krupp, J. / Stenmark, P.
Funding support Sweden, 1items
OrganizationGrant numberCountry
Swedish Research Council Sweden
CitationJournal: Sci Adv / Year: 2019
Title: Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models.
Authors: Elliott, M. / Favre-Guilmard, C. / Liu, S.M. / Maignel, J. / Masuyer, G. / Beard, M. / Boone, C. / Carre, D. / Kalinichev, M. / Lezmi, S. / Mir, I. / Nicoleau, C. / Palan, S. / Perier, C. / ...Authors: Elliott, M. / Favre-Guilmard, C. / Liu, S.M. / Maignel, J. / Masuyer, G. / Beard, M. / Boone, C. / Carre, D. / Kalinichev, M. / Lezmi, S. / Mir, I. / Nicoleau, C. / Palan, S. / Perier, C. / Raban, E. / Zhang, S. / Dong, M. / Stenmark, P. / Krupp, J.
History
DepositionMar 29, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 16, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.0Sep 7, 2022Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Other / Polymer sequence / Structure summary
Category: atom_site / database_2 ...atom_site / database_2 / entity_poly / pdbx_database_status / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / struct_conf / struct_ref_seq
Item: _atom_site.auth_asym_id / _atom_site.auth_seq_id ..._atom_site.auth_asym_id / _atom_site.auth_seq_id / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_poly.pdbx_strand_id / _pdbx_database_status.pdb_format_compatible / _pdbx_nonpoly_scheme.pdb_strand_id / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_poly_seq_scheme.pdb_strand_id / _pdbx_unobs_or_zero_occ_residues.auth_asym_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_validate_torsion.auth_seq_id / _struct_conf.beg_auth_asym_id / _struct_conf.beg_auth_seq_id / _struct_conf.end_auth_asym_id / _struct_conf.end_auth_seq_id / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.pdbx_strand_id
Revision 2.1Jan 31, 2024Group: 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 type B
C: Synaptotagmin-1
B: Botulinum neurotoxin type B
33: Synaptotagmin-1


Theoretical massNumber of molelcules
Total (without water)115,2584
Polymers115,2584
Non-polymers00
Water7,909439
1
A: Botulinum neurotoxin type B
C: Synaptotagmin-1


  • defined by author&software
  • Evidence: gel filtration, Binding domain of botulinum toxin B is a monomer in solution
  • 57.6 kDa, 2 polymers
Theoretical massNumber of molelcules
Total (without water)57,6292
Polymers57,6292
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1350 Å2
ΔGint-9 kcal/mol
Surface area20440 Å2
MethodPISA
2
B: Botulinum neurotoxin type B
33: Synaptotagmin-1


  • defined by author&software
  • 57.6 kDa, 2 polymers
Theoretical massNumber of molelcules
Total (without water)57,6292
Polymers57,6292
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1260 Å2
ΔGint-9 kcal/mol
Surface area20330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)48.300, 212.790, 52.910
Angle α, β, γ (deg.)90.00, 91.08, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Botulinum neurotoxin type B / BoNT/B / Bontoxilysin-B


Mass: 55157.051 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Binding domain of Botulinum Neurotoxin type B expressed with a N-terminal poly-His tag
Source: (gene. exp.) Clostridium botulinum (bacteria) / Gene: botB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P10844, bontoxilysin
#2: Protein/peptide Synaptotagmin-1 / / Synaptotagmin I / SytI / p65


Mass: 2471.825 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Synthetic peptide corresponding to human synaptotagmin 1 residues [33-53]
Source: (synth.) Homo sapiens (human) / References: UniProt: P21579
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 439 / 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: 2.5 Å3/Da / Density % sol: 50.5 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M Amino acids 0.1 M Buffer System 2 7.5 50 % v/v Precipitant Mix 1

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 24, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2→70.93 Å / Num. obs: 71658 / % possible obs: 99.9 % / Redundancy: 6.9 % / CC1/2: 0.998 / Rmerge(I) obs: 0.103 / Rpim(I) all: 0.065 / Rrim(I) all: 0.122 / Net I/σ(I): 9.6
Reflection shellResolution: 2→2.05 Å / Redundancy: 7 % / Rmerge(I) obs: 1.31 / Num. unique obs: 5300 / CC1/2: 0.681 / Rpim(I) all: 0.815 / Rrim(I) all: 1.546 / % possible all: 99.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0222refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4KBB

4kbb


Resolution: 2→53.25 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.941 / SU B: 6.198 / SU ML: 0.156 / Cross valid method: THROUGHOUT / ESU R: 0.184 / ESU R Free: 0.164 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23545 3555 5 %RANDOM
Rwork0.19361 ---
obs0.19568 68048 99.86 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 43.992 Å2
Baniso -1Baniso -2Baniso -3
1--1.8 Å20 Å2-0.73 Å2
2---2.41 Å20 Å2
3---4.24 Å2
Refinement stepCycle: 1 / Resolution: 2→53.25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7455 0 0 439 7894
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0147659
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176785
X-RAY DIFFRACTIONr_angle_refined_deg1.0891.65110313
X-RAY DIFFRACTIONr_angle_other_deg0.7851.64215909
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.2945874
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.14923.609460
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.509151435
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.2311537
X-RAY DIFFRACTIONr_chiral_restr0.0550.2976
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.028485
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021563
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.8254.3093511
X-RAY DIFFRACTIONr_mcbond_other2.8234.3083510
X-RAY DIFFRACTIONr_mcangle_it4.5276.4414380
X-RAY DIFFRACTIONr_mcangle_other4.5286.4424381
X-RAY DIFFRACTIONr_scbond_it2.9794.6714148
X-RAY DIFFRACTIONr_scbond_other2.9794.6734149
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.7996.8595934
X-RAY DIFFRACTIONr_long_range_B_refined8.26547.818595
X-RAY DIFFRACTIONr_long_range_B_other8.25547.6588521
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2→2.052 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.343 238 -
Rwork0.333 5058 -
obs--99.72 %

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