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- PDB-3k3q: Crystal Structure of a Llama Antibody complexed with the C. Botul... -

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Basic information

Entry
Database: PDB / ID: 3k3q
TitleCrystal Structure of a Llama Antibody complexed with the C. Botulinum Neurotoxin Serotype A Catalytic Domain
Components
  • (Botulinum neurotoxin type A) x 2
  • llama Aa1 VHH domain
KeywordsIMMUNE SYSTEM / llama / VHH / antibody / botulinum / neurotoxin / BoNT / Cell junction / Cell membrane / Cytoplasm / Disulfide bond / Hydrolase / Membrane / Metal-binding / Metalloprotease / Pharmaceutical / Protease / Secreted / Synapse / Toxin / Transmembrane / Zinc
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 / membrane => GO:0016020 / metalloendopeptidase activity / toxin activity ...host cell junction / negative regulation of neurotransmitter secretion / bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / membrane => GO:0016020 / 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 / Neutral zinc metallopeptidases, zinc-binding region signature. / Few Secondary Structures / Irregular / Concanavalin A-like lectin/glucanase domain superfamily / Immunoglobulins / Alpha-Beta Complex / Up-down Bundle / Immunoglobulin-like / Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Botulinum neurotoxin type A / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesLama glama (llama)
Clostridium botulinum A str. Hall (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.6 Å
AuthorsThompson, A.A. / Dong, J. / Marks, J.D. / Stevens, R.C.
CitationJournal: J.Mol.Biol. / Year: 2010
Title: A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic alpha-Exosite Binding Region.
Authors: Dong, J. / Thompson, A.A. / Fan, Y. / Lou, J. / Conrad, F. / Ho, M. / Pires-Alves, M. / Wilson, B.A. / Stevens, R.C. / Marks, J.D.
History
DepositionOct 4, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 1, 2017Group: Refinement description / Category: software
Revision 1.3Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entry_details.has_protein_modification / _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_ref_seq_dif.details / _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: llama Aa1 VHH domain
B: Botulinum neurotoxin type A
C: Botulinum neurotoxin type A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,1954
Polymers65,1293
Non-polymers651
Water1,26170
1
A: llama Aa1 VHH domain
B: Botulinum neurotoxin type A
C: Botulinum neurotoxin type A
hetero molecules

A: llama Aa1 VHH domain
B: Botulinum neurotoxin type A
C: Botulinum neurotoxin type A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,3908
Polymers130,2596
Non-polymers1312
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y,-z1
Buried area21780 Å2
ΔGint-223 kcal/mol
Surface area45440 Å2
MethodPISA
2


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9020 Å2
ΔGint-95 kcal/mol
Surface area24590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)168.282, 48.764, 103.643
Angle α, β, γ (deg.)90.000, 116.750, 90.000
Int Tables number5
Space group name H-MC121
DetailsAuthors state it is possible that a hexamer exists in solution, but this would be in equilibrium with the trimer.

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Components

#1: Protein llama Aa1 VHH domain


Mass: 16147.911 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Gene: llama VHH immunoglobulin / Plasmid: pET20b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3)
#2: Protein Botulinum neurotoxin type A / BoNT/A / Bontoxilysin-A / BOTOX / Botulinum neurotoxin A light chain


Mass: 28359.986 Da / Num. of mol.: 1
Fragment: N-terminal fragment of BoNT catalytic domain (residues 3-250)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A str. Hall (bacteria)
Gene: botA, CBO0806, CLC_0862, neurotoxin catalytic domain / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3)
References: UniProt: A5HZZ9, UniProt: P0DPI1*PLUS, bontoxilysin
#3: Protein Botulinum neurotoxin type A / BoNT/A / Bontoxilysin-A / BOTOX / Botulinum neurotoxin A light chain


Mass: 20621.465 Da / Num. of mol.: 1
Fragment: C-terminal fragment of BoNT catalytic domain (residues 251-425)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A str. Hall (bacteria)
Gene: botA, CBO0806, CLC_0862, neurotoxin catalytic domain / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3)
References: UniProt: A5HZZ9, UniProt: P0DPI1*PLUS, bontoxilysin
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 70 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY
Sequence detailsTHERE IS A VAL -> ALA NATURAL VARIANT AT RESIDUE 27.

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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: 2.92 Å3/Da / Density % sol: 57.81 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 25% ethylene glycol, pH 7.0, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 93 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Oct 16, 2008
RadiationMonochromator: Double crystal cryo-cooled Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionRedundancy: 2.8 % / Av σ(I) over netI: 7.33 / Number: 64075 / Rmerge(I) obs: 0.117 / Χ2: 1 / D res high: 2.6 Å / D res low: 40 Å / Num. obs: 22641 / % possible obs: 96.5
Diffraction reflection shell
Highest resolution (Å)Lowest resolution (Å)% possible obs (%)IDRmerge(I) obsChi squaredRedundancy
7.054095.410.0640.9543
5.67.0597.710.0680.9833.1
4.895.698.110.0681.0023.1
4.444.8998.410.0780.9933.1
4.134.4498.810.0890.983.1
3.884.139910.0980.9763.1
3.693.8899.110.1230.9943.1
3.533.6999.210.1561.0093.1
3.393.5399.310.1580.9933.1
3.283.3999.310.1690.9943.1
3.173.2899.510.2131.023
3.083.1799.110.221.0152.9
33.0898.510.2521.012.9
2.93397.610.2741.0032.7
2.862.9396.510.270.9862.6
2.82.8695.110.3081.0122.5
2.742.893.610.3160.9992.3
2.692.7490.810.330.9892.2
2.642.6990.110.3811.0062.1
2.62.6483.910.3141.0031.9
ReflectionResolution: 2.6→40 Å / Num. obs: 22641 / % possible obs: 96.5 % / Redundancy: 2.8 % / Biso Wilson estimate: 26.3 Å2 / Rmerge(I) obs: 0.117 / Χ2: 0.995 / Net I/σ(I): 9.5
Reflection shellResolution: 2.6→2.64 Å / Redundancy: 1.9 % / Rmerge(I) obs: 0.314 / Mean I/σ(I) obs: 2.16 / Num. unique all: 977 / Χ2: 1.003 / % possible all: 83.9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
CNSrefinement
PDB_EXTRACT3.005data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→40 Å / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.823 / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.256 1029 4.4 %random
Rwork0.218 ---
obs-21269 90.4 %-
Solvent computationBsol: 20.541 Å2
Displacement parametersBiso max: 91.81 Å2 / Biso mean: 41.719 Å2 / Biso min: 3.39 Å2
Baniso -1Baniso -2Baniso -3
1--13.245 Å20 Å2-2.481 Å2
2---1.28 Å20 Å2
3---14.525 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.43 Å0.34 Å
Luzzati d res low-5 Å
Luzzati sigma a0.48 Å0.48 Å
Refinement stepCycle: LAST / Resolution: 2.6→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4287 0 1 70 4358
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_mcbond_it1.4691.5
X-RAY DIFFRACTIONc_scbond_it1.9922
X-RAY DIFFRACTIONc_mcangle_it2.5652
X-RAY DIFFRACTIONc_scangle_it3.1572.5
Xplor file
Refine-IDSerial noParam file
X-RAY DIFFRACTION1CNS_TOPPAR:protein_rep.param
X-RAY DIFFRACTION2CNS_TOPPAR:water_rep.param
X-RAY DIFFRACTION3CNS_TOPPAR:ion.param

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