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- EMDB-22699: Structure of TyTx1 Fab in Complex with Typhoid Toxin -

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Basic information

Entry
Database: EMDB / ID: EMD-22699
TitleStructure of TyTx1 Fab in Complex with Typhoid Toxin
Map dataFull map
Sample
  • Complex: Complex of TyTx1 Fab with Typhoid toxin
    • Complex: Typhoid toxin
      • Protein or peptide: Pertussis like toxin subunit B
      • Protein or peptide: Pertussis toxin-like subunit ArtA
    • Complex: TyTx1 Fab
      • Protein or peptide: Fab Light Chain Variable Domain
      • Protein or peptide: Fab Heavy Chain Variable Domain
Function / homology
Function and homology information


: / NAD+ ADP-ribosyltransferase activity / extracellular region
Similarity search - Function
Bordetella pertussis toxin A / Pertussis toxin, subunit 1 / Bordetella pertussis toxin B, subunit 2/3, C-terminal / Pertussis toxin, subunit 2 and 3, C-terminal domain / Enterotoxin
Similarity search - Domain/homology
Pertussis like toxin subunit B / Pertussis toxin-like subunit ArtA
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhi (bacteria) / Mus musculus (house mouse) / Salmonella enterica subsp. enterica serovar Typhi str. CT18 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsNguyen T / Feathers JR / Fromme JC / Song J
Funding support United States, 3 items
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 Allergy and Infectious Diseases (NIH/NIAID)R01 GM098621 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)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, 2020-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateSep 22, 2021-
Current statusSep 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0102
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0102
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k7h
  • Surface level: 0.0102
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22699.png

Downloads & links

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Map

FileDownload / File: emd_22699.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0102 / Movie #1: 0.0102
Minimum - Maximum-0.0350301 - 0.058804728
Average (Standard dev.)-1.4970027e-06 (±0.0014786812)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z249.000249.000249.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0350.059-0.000

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Supplemental data

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Additional map: Post-processed map

Fileemd_22699_additional_1.map
AnnotationPost-processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed Masked Map

Fileemd_22699_additional_2.map
AnnotationPost-processed Masked Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_22699_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_22699_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of TyTx1 Fab with Typhoid toxin

EntireName: Complex of TyTx1 Fab with Typhoid toxin
Components
  • Complex: Complex of TyTx1 Fab with Typhoid toxin
    • Complex: Typhoid toxin
      • Protein or peptide: Pertussis like toxin subunit B
      • Protein or peptide: Pertussis toxin-like subunit ArtA
    • Complex: TyTx1 Fab
      • Protein or peptide: Fab Light Chain Variable Domain
      • Protein or peptide: Fab Heavy Chain Variable Domain

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Supramolecule #1: Complex of TyTx1 Fab with Typhoid toxin

SupramoleculeName: Complex of TyTx1 Fab with Typhoid toxin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragment generated by proteolytic cleavage of IgG antibody in complex with purified Typhoid toxin

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Supramolecule #2: Typhoid toxin

SupramoleculeName: Typhoid toxin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #4
Details: S. Typhi A2B5 toxin pltA-E133A cdtB-H160Q double mutant (catalytically inactivated toxin)
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: TyTx1 Fab

SupramoleculeName: TyTx1 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Fab fragment generated by proteolytic cleavage of IgG antibody TyTx1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse) / Recombinant cell: Hybridoma

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Macromolecule #1: Pertussis like toxin subunit B

MacromoleculeName: Pertussis like toxin subunit B / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi str. CT18 (bacteria)
Molecular weightTheoretical: 12.563042 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
EWTGDNTNAY YSDEVISELH VGQIDTSPYF CIKTVKANGS GTPVVACAVS KQSIWAPSFK ELLDQARYFY STGQSVRIHV QKNIWTYPL FVNTFSANAL VGLSSCSATQ CFGPK

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Macromolecule #2: Fab Light Chain Variable Domain

MacromoleculeName: Fab Light Chain Variable Domain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.679397 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DIVMSQSPSS LAVSAGEKVN MSCKSSQSLF NSRTRKNHLA WYQQKPGQSP KLMIYWASTG ECVVRDRFTG SGCGTDFTLT ISSVQDEDR AVYLCKQSHN RALTFGCGTK LEMKR

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Macromolecule #3: Fab Heavy Chain Variable Domain

MacromoleculeName: Fab Heavy Chain Variable Domain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.802131 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
EIQSQQCGPE LVKPGSSVKV SCKASGYAFT NYKALGSKQS HGKSLEWIGY IDPYNSDSSY NQQFKDKATL TVDKSSSTAY MYLNSLTSE DSAVYYCAGL ELTGTLPYWG QGTLVTVSA

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Macromolecule #4: Pertussis toxin-like subunit ArtA

MacromoleculeName: Pertussis toxin-like subunit ArtA / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi str. CT18 (bacteria)
Molecular weightTheoretical: 1.66597 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
FYDARPVIEL ILSK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
15.0 mMTristris(hydroxymethyl)aminomethane
150.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 2.5 second before plunging.
DetailsFreshly prepared size-exclusion-chromatography purified complex of TyTx1 Fab and Typhoid toxin catalytically inactivated toxin double mutant.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 49000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4k6l

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 326766
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4rhr


1mhh

chain_id: A

4h20

chain_id: H
DetailsInitial 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.
Output model

PDB-7k7h:
Density-fitted Model Structure of Antibody Variable Domains of TyTx1 in Complex with PltB pentamer of Typhoid Toxin

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