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- PDB-7k7i: Density-fitted Model Structure of Antibody Variable Domains of Ty... -

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Basic information

Entry
Database: PDB / ID: 7k7i
TitleDensity-fitted Model Structure of Antibody Variable Domains of TyTx4 in Complex with PltB pentamer of Typhoid Toxin
Components
  • Fab Heavy Chain Variable Domain
  • Fab Light Chain Variable Domain
  • Putative pertussis-like toxin subunit
KeywordsTOXIN / Typhoid Toxin / A2B5 / Antibody / Fab
Function / homologyBordetella pertussis toxin B, subunit 2/3, C-terminal / Pertussis toxin, subunit 2 and 3, C-terminal domain / Enterotoxin / Immunoglobulins / Immunoglobulin-like / Sandwich / extracellular region / Mainly Beta / Pertussis-like toxin subunit
Function and homology information
Biological speciesSalmonella typhi (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsNguyen, T. / Feathers, J.R. / Fromme, J.C. / Song, J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R03 AI135767 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM098621 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM116942 United States
CitationJournal: Cell Rep / Year: 2021
Title: The structural basis of Salmonella AB toxin neutralization by antibodies targeting the glycan-receptor binding subunits.
Authors: Tri Nguyen / Angelene F Richards / Durga P Neupane / J Ryan Feathers / Yi-An Yang / Ji Hyun Sim / Haewon Byun / Sohyoung Lee / Changhwan Ahn / Greta Van Slyke / J Christopher Fromme / ...Authors: Tri Nguyen / Angelene F Richards / Durga P Neupane / J Ryan Feathers / Yi-An Yang / Ji Hyun Sim / Haewon Byun / Sohyoung Lee / Changhwan Ahn / Greta Van Slyke / J Christopher Fromme / Nicholas J Mantis / Jeongmin Song /
Abstract: Many bacterial pathogens secrete AB toxins comprising two functionally distinct yet complementary "A" and "B" subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits ...Many bacterial pathogens secrete AB toxins comprising two functionally distinct yet complementary "A" and "B" subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits recognize specific sets of host glycans to deliver the toxin into target host cells. Here, we offer the molecular mechanism by which neutralizing antibodies, which have the potential to bind to all glycan-receptor binding sites and thus completely inhibit toxin binding to host cells, are inhibited from exerting this action. Cryogenic electron microscopy (cryo-EM)-based analyses indicate that the skewed positioning of the toxin A subunit(s) toward one side of the toxin B pentamer inhibited neutralizing antibody binding to the laterally located epitopes, rendering some glycan-receptor binding sites that remained available for the toxin binding and endocytosis process, which is strikingly different from the counterpart antibodies recognizing the far side-located epitopes. These results highlight additional features of the toxin-antibody interactions and offer important insights into anti-toxin strategies.
History
DepositionSep 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 22, 2021Group: Database references / Category: citation / citation_author
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 / _citation_author.name
Revision 1.2Nov 20, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-22700
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  • Superimposition on EM map
  • EMDB-22700
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Putative pertussis-like toxin subunit
B: Putative pertussis-like toxin subunit
C: Putative pertussis-like toxin subunit
D: Putative pertussis-like toxin subunit
E: Putative pertussis-like toxin subunit
L: Fab Light Chain Variable Domain
H: Fab Heavy Chain Variable Domain
K: Fab Light Chain Variable Domain
J: Fab Heavy Chain Variable Domain
N: Fab Light Chain Variable Domain
M: Fab Heavy Chain Variable Domain
Q: Fab Light Chain Variable Domain
P: Fab Heavy Chain Variable Domain
Y: Fab Light Chain Variable Domain
X: Fab Heavy Chain Variable Domain


Theoretical massNumber of molelcules
Total (without water)187,38315
Polymers187,38315
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Putative pertussis-like toxin subunit / Subtilase cytotoxin subunit B / PltB


Mass: 12563.042 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhi (bacteria)
Gene: STY1891, t1107, ABP05_22510, AXU46_23480, E2E79_23180, E2E83_22990, E2F00_23020, E2F08_23180, E2F09_23155, E2F13_22745, PltB
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8Z6A3
#2: Antibody
Fab Light Chain Variable Domain


Mass: 11753.811 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody
Fab Heavy Chain Variable Domain


Mass: 13159.710 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of TyTx4 Fab with Typhoid Toxin Catalytic Inactivated MutantCOMPLEXMap of Fab fragment of proteolytically cleaved IgG antibody TyTx4 in complex with purified Typhoid toxin PltA E133A and CdtB H160Q double mutantall0MULTIPLE SOURCES
2Typhoid Toxin Catalytically Inactivated MutantCOMPLEXS. Typhi A2B5 toxin PltA E133A & CdtB H160Q double mutant#11RECOMBINANT
3TyTx4 FabCOMPLEXFab fragment generated by proteolytic cleavage of IgG antibody TyTx4#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Salmonella enterica subsp. enterica serovar Typhi (bacteria)90370
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Escherichia coli BL21(DE3) (bacteria)469008
23Mus musculus (house mouse)10090Hybridoma
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
115 mMtris(hydroxymethyl)aminomethaneTris1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Freshly prepared size-exclusion-chromatography purified complex between Fab fragment of TyTx4 and Typhoid toxin wild-type
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: blot for 2.0 second before plunging

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 49000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 51.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9PHENIX1.16-3549model refinement
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97594 / Symmetry type: POINT
Atomic model buildingDetails: Initial local fitting was done using Chimera and then Coot was used for rebuilding Fab variable domains into correct sequences. Refinement was performed using Real Space Refine in PHENIX and ...Details: Initial local fitting was done using Chimera and then Coot was used for rebuilding Fab variable domains into correct sequences. Refinement was performed using Real Space Refine in PHENIX and was iterated with manual building in Coot.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDSource nameType
14RHR

4rhr

4RHR1PDBexperimental model
22OZ4

2oz4

L2OZ42PDBexperimental model
34M48

4m48

H4M483PDBexperimental model
4
5
6
7

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