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- PDB-6g5f: Crystal structure of an engineered Botulinum Neurotoxin type B mu... -

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Basic information

Entry
Database: PDB / ID: 6g5f
TitleCrystal structure of an engineered Botulinum Neurotoxin type B mutant E1191M/S1199Y 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


Toxicity of botulinum toxin type G (botG) / clathrin-sculpted acetylcholine transport vesicle membrane / clathrin-sculpted glutamate transport vesicle membrane / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / spontaneous neurotransmitter secretion / regulation of regulated secretory pathway / Toxicity of botulinum toxin type B (botB) / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane ...Toxicity of botulinum toxin type G (botG) / clathrin-sculpted acetylcholine transport vesicle membrane / clathrin-sculpted glutamate transport vesicle membrane / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / spontaneous neurotransmitter secretion / regulation of regulated secretory pathway / Toxicity of botulinum toxin type B (botB) / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane / calcium-dependent activation of synaptic vesicle fusion / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / chromaffin granule membrane / GABA synthesis, release, reuptake and degradation / Acetylcholine Neurotransmitter Release Cycle / clathrin-sculpted monoamine transport vesicle membrane / calcium ion sensor activity / regulation of calcium ion-dependent exocytosis / Serotonin Neurotransmitter Release Cycle / exocytic vesicle / Norepinephrine Neurotransmitter Release Cycle / Dopamine Neurotransmitter Release Cycle / vesicle organization / protein heterooligomerization / positive regulation of dendrite extension / vesicle docking / regulation of exocytosis / Glutamate Neurotransmitter Release Cycle / positive regulation of dopamine secretion / bontoxilysin / vesicle fusion / dense core granule / calcium-dependent phospholipid binding / neuron projection terminus / membraneless organelle assembly / host cell presynaptic membrane / host cell cytoplasmic vesicle / neurotransmitter secretion / presynaptic active zone / Neurexins and neuroligins / syntaxin-1 binding / host cell cytosol / low-density lipoprotein particle receptor binding / clathrin binding / phosphatidylserine binding / regulation of synaptic vesicle exocytosis / excitatory synapse / protein transmembrane transporter activity / detection of calcium ion / positive regulation of synaptic transmission / postsynaptic cytosol / regulation of synaptic transmission, glutamatergic / presynaptic cytosol / vesicle-mediated transport / phosphatidylinositol-4,5-bisphosphate binding / cellular response to calcium ion / hippocampal mossy fiber to CA3 synapse / SNARE binding / molecular condensate scaffold activity / clathrin-coated endocytic vesicle membrane / metalloendopeptidase activity / calcium-dependent protein binding / synaptic vesicle / synaptic vesicle membrane / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / toxin activity / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / cell differentiation / calmodulin binding / neuron projection / postsynaptic density / protein heterodimerization activity / axon / calcium ion binding / lipid binding / host cell plasma membrane / glutamatergic synapse / Golgi apparatus / proteolysis / extracellular region / zinc ion binding / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Synaptotagmin / 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 ...Synaptotagmin / 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 / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
MALONATE ION / Botulinum neurotoxin type B / Synaptotagmin-1
Similarity search - Component
Biological speciesClostridium botulinum (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 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 / Database references / Derived calculations / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / database_2 / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / struct_conf / struct_ref_seq
Item: _atom_site.auth_seq_id / _atom_site_anisotrop.pdbx_auth_seq_id ..._atom_site.auth_seq_id / _atom_site_anisotrop.pdbx_auth_seq_id / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _struct_conf.beg_auth_seq_id / _struct_conf.end_auth_seq_id / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end
Revision 2.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 2.2Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Botulinum neurotoxin type B
B: Botulinum neurotoxin type B
P: Synaptotagmin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,9847
Polymers304,6053
Non-polymers3784
Water4,828268
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13260 Å2
ΔGint-53 kcal/mol
Surface area57530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)176.078, 356.419, 103.939
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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


Mass: 151066.797 Da / Num. of mol.: 2 / Mutation: E231Q, H234Y
Source method: isolated from a genetically manipulated source
Details: Catalytic domain of an engineered inactive (mutations E231Q/H234Y) botulinum toxin type B mutant E1191M/S1199Y. Please note the protein is expressed as a single polypeptide which is post- ...Details: Catalytic domain of an engineered inactive (mutations E231Q/H234Y) botulinum toxin type B mutant E1191M/S1199Y. Please note the protein is expressed as a single polypeptide which is post-translationally cleaved into a di-chain molecule (light and heavy chains - chain A and B)
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.: 1 / Source method: obtained synthetically
Details: Synthetic peptide corresponding to human synaptotagmin 1 residues [33-53]
Source: (synth.) Homo sapiens (human) / References: UniProt: P21579
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-MLI / MALONATE ION


Mass: 102.046 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H2O4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 268 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 5.44 Å3/Da / Density % sol: 77.4 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 1.1 M sodium malonate dibasic monohydrate, HEPES pH 7.0, 0.5% v/v Jeffamine ED-2003

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.928 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 1, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.928 Å / Relative weight: 1
ReflectionResolution: 2.5→98.5 Å / Num. obs: 113032 / % possible obs: 100 % / Redundancy: 7.1 % / CC1/2: 0.992 / Rmerge(I) obs: 0.1 / Rpim(I) all: 0.061 / Rrim(I) all: 0.117 / Net I/σ(I): 11
Reflection shellResolution: 2.5→2.54 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.947 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 5535 / CC1/2: 0.492 / Rpim(I) all: 0.598 / Rrim(I) all: 1.124 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0158refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1EPW

1epw


Resolution: 2.5→98.5 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.94 / SU B: 18.852 / SU ML: 0.186 / Cross valid method: THROUGHOUT / ESU R: 0.214 / ESU R Free: 0.189 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.23182 5577 4.9 %RANDOM
Rwork0.20173 ---
obs0.2032 107431 99.99 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 73.562 Å2
Baniso -1Baniso -2Baniso -3
1-2.04 Å20 Å20 Å2
2---0.12 Å20 Å2
3----1.92 Å2
Refinement stepCycle: 1 / Resolution: 2.5→98.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10683 0 25 268 10976
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0210960
X-RAY DIFFRACTIONr_bond_other_d0.0010.029981
X-RAY DIFFRACTIONr_angle_refined_deg1.1711.95614805
X-RAY DIFFRACTIONr_angle_other_deg0.876323263
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.05351304
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.73425.502558
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.584151983
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.3641535
X-RAY DIFFRACTIONr_chiral_restr0.070.21602
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.0212107
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022220
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_it1.495.3035227
X-RAY DIFFRACTIONr_mcbond_other1.495.3035226
X-RAY DIFFRACTIONr_mcangle_it2.6787.9486526
X-RAY DIFFRACTIONr_mcangle_other2.6787.9496527
X-RAY DIFFRACTIONr_scbond_it1.2155.4035733
X-RAY DIFFRACTIONr_scbond_other1.2135.4025731
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.2088.0378279
X-RAY DIFFRACTIONr_long_range_B_refined4.38757.85911542
X-RAY DIFFRACTIONr_long_range_B_other4.36757.79811520
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.5→2.565 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.357 396 -
Rwork0.366 7853 -
obs--99.98 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.28570.1119-0.04251.14360.21460.6043-0.04160.0159-0.0843-0.0423-0.00790.0977-0.0361-0.08050.04950.33770.03810.00310.0151-0.00830.040769.347829.3293-5.9533
20.5081-0.2729-0.13210.4558-0.00320.16220.0277-0.01660.1421-0.06120.0442-0.0926-0.1377-0.0584-0.07190.60.1160.02380.03810.00770.05762.794980.935-0.7479
33.48242.22144.94732.5921.30289.95230.27620.02460.06430.5934-0.12490.2022-0.22950.2451-0.15120.65680.12040.16090.1787-0.18790.429645.3146123.889831.2088
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 441
2X-RAY DIFFRACTION2B442 - 1291
3X-RAY DIFFRACTION3P43 - 60

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