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- PDB-6g5g: Crystal structure of an engineered Botulinum Neurotoxin type B mu... -

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Basic information

Entry
Database: PDB / ID: 6g5g
TitleCrystal structure of an engineered Botulinum Neurotoxin type B mutant E1191M/S1199Y in complex with human synaptotagmin 2
Components
  • Botulinum neurotoxin type B
  • Synaptotagmin-2
KeywordsTOXIN / botulinum toxin / neurotoxin / protein engineering / receptor binding
Function / homology
Function and homology information


Toxicity of botulinum toxin type B (botB) / chromaffin granule membrane / inositol 1,3,4,5 tetrakisphosphate binding / dense core granule / calcium ion-regulated exocytosis of neurotransmitter / exocytic vesicle / bontoxilysin / positive regulation of dendrite extension / host cell presynaptic membrane / host cell cytoplasmic vesicle ...Toxicity of botulinum toxin type B (botB) / chromaffin granule membrane / inositol 1,3,4,5 tetrakisphosphate binding / dense core granule / calcium ion-regulated exocytosis of neurotransmitter / exocytic vesicle / bontoxilysin / positive regulation of dendrite extension / host cell presynaptic membrane / host cell cytoplasmic vesicle / calcium-dependent phospholipid binding / Neurexins and neuroligins / host cell cytosol / clathrin binding / phosphatidylserine binding / protein transmembrane transporter activity / synaptic vesicle endocytosis / SNARE binding / clathrin-coated endocytic vesicle membrane / metalloendopeptidase activity / synaptic vesicle membrane / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / toxin activity / cell differentiation / axon / lipid binding / calcium ion binding / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / membrane / plasma membrane
Similarity search - Function
Synaptotagmin / 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 C-terminal / Clostridium neurotoxin, receptor binding N-terminal ...Synaptotagmin / 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 C-terminal / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, C-terminal receptor binding / 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
ACETATE ION / DI(HYDROXYETHYL)ETHER / Botulinum neurotoxin type B / Synaptotagmin-2
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 1.2Aug 11, 2021Group: Database references / Derived calculations ...Database references / Derived calculations / Source and taxonomy / Structure summary
Category: database_2 / entity ...database_2 / entity / entity_name_com / pdbx_entity_src_syn / pdbx_struct_conn_angle / struct_conn / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.details / _entity.pdbx_description / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_common_name / _pdbx_entity_src_syn.organism_scientific / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.pdbx_db_accession
Revision 1.3Jan 17, 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

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Assembly

Deposited unit
A: Botulinum neurotoxin type B
B: Botulinum neurotoxin type B
P: Synaptotagmin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)305,48513
Polymers304,6363
Non-polymers84810
Water14,196788
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13210 Å2
ΔGint-51 kcal/mol
Surface area56210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)166.600, 211.050, 120.320
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11B-1914-

HOH

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Components

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Protein / Protein/peptide , 2 types, 3 molecules ABP

#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-2 / Synaptotagmin II / SytII


Mass: 2502.836 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Synthetic peptide corresponding to human synaptotagmin 2 residues [37-57]
Source: (synth.) Homo sapiens (human) / References: UniProt: Q8N9I0

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Non-polymers , 7 types, 798 molecules

#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#6: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#7: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#8: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 788 / 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: 3.5 Å3/Da / Density % sol: 65.1 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.2 M magnesium chloride, Tris pH 7.0, 10% w/v PEG 8000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: May 5, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2→57.5 Å / Num. obs: 142330 / % possible obs: 99.9 % / Redundancy: 6.6 % / CC1/2: 0.998 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.071 / Rrim(I) all: 0.134 / Net I/σ(I): 9.8
Reflection shellResolution: 2→2.03 Å / Redundancy: 6.7 % / Rmerge(I) obs: 1.399 / Num. unique obs: 7015 / CC1/2: 0.499 / Rpim(I) all: 0.866 / Rrim(I) all: 1.652 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0218refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1EPW

1epw


Resolution: 2→57.5 Å / Cor.coef. Fo:Fc: 0.964 / Cor.coef. Fo:Fc free: 0.947 / SU B: 4.802 / SU ML: 0.122 / Cross valid method: THROUGHOUT / ESU R: 0.139 / ESU R Free: 0.133 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22339 7218 5.1 %RANDOM
Rwork0.18962 ---
obs0.19133 135070 99.91 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 44.844 Å2
Baniso -1Baniso -2Baniso -3
1-1.61 Å20 Å20 Å2
2---2.65 Å20 Å2
3---1.05 Å2
Refinement stepCycle: 1 / Resolution: 2→57.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10673 0 55 788 11516
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0210986
X-RAY DIFFRACTIONr_bond_other_d0.0010.0210061
X-RAY DIFFRACTIONr_angle_refined_deg1.2511.95814824
X-RAY DIFFRACTIONr_angle_other_deg0.729323469
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.27151294
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.71825.503567
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.346152016
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.2061536
X-RAY DIFFRACTIONr_chiral_restr0.0720.21595
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0212081
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022227
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_it2.5564.3835181
X-RAY DIFFRACTIONr_mcbond_other2.5564.3825178
X-RAY DIFFRACTIONr_mcangle_it4.0686.5596472
X-RAY DIFFRACTIONr_mcangle_other4.0686.5596473
X-RAY DIFFRACTIONr_scbond_it2.924.6765805
X-RAY DIFFRACTIONr_scbond_other2.9174.6775805
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.7386.8778353
X-RAY DIFFRACTIONr_long_range_B_refined7.05550.88812701
X-RAY DIFFRACTIONr_long_range_B_other6.9950.65212531
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
Rfree0.331 529 -
Rwork0.334 9888 -
obs--99.9 %

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