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- EMDB-22700: Density of TyTx4 Fab in Complex with Typhoid Toxin -

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Basic information

Entry
Database: EMDB / ID: EMD-22700
TitleDensity of TyTx4 Fab in Complex with Typhoid Toxin
Map dataFull-map
Sample
  • Complex: Complex of TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant
    • Complex: Typhoid Toxin Catalytically Inactivated Mutant
      • Protein or peptide: Putative pertussis-like toxin subunit
    • Complex: TyTx4 Fab
      • Protein or peptide: Fab Light Chain Variable Domain
      • Protein or peptide: Fab Heavy Chain Variable Domain
Function / homologyBordetella pertussis toxin B, subunit 2/3, C-terminal / Pertussis toxin, subunit 2 and 3, C-terminal domain / Enterotoxin / extracellular region / Pertussis-like toxin subunit
Function and homology information
Biological speciesSalmonella enterica subsp. enterica serovar Typhi (bacteria) / Mus musculus (house mouse) / Salmonella typhi (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 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 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, 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: 0.00845
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00845
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k7i
  • Surface level: 0.00845
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7k7i
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22700.png

Downloads & links

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Map

FileDownload / File: emd_22700.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull-map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.00845 / Movie #1: 0.00845
Minimum - Maximum-0.009904241 - 0.038402427
Average (Standard dev.)8.94757e-06 (±0.0009573523)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z332.000332.000332.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0100.0380.000

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Supplemental data

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Additional map: Post-processed Map

Fileemd_22700_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_22700_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_22700_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_22700_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 TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant

EntireName: Complex of TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant
Components
  • Complex: Complex of TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant
    • Complex: Typhoid Toxin Catalytically Inactivated Mutant
      • Protein or peptide: Putative pertussis-like toxin subunit
    • Complex: TyTx4 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 TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant

SupramoleculeName: Complex of TyTx4 Fab with Typhoid Toxin Catalytic Inactivated Mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Map of Fab fragment of proteolytically cleaved IgG antibody TyTx4 in complex with purified Typhoid toxin PltA E133A and CdtB H160Q double mutant

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Supramolecule #2: Typhoid Toxin Catalytically Inactivated Mutant

SupramoleculeName: Typhoid Toxin Catalytically Inactivated Mutant / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: S. Typhi A2B5 toxin PltA E133A & CdtB H160Q double mutant
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: TyTx4 Fab

SupramoleculeName: TyTx4 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Fab fragment generated by proteolytic cleavage of IgG antibody TyTx4
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse) / Recombinant cell: Hybridoma

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Macromolecule #1: Putative pertussis-like toxin subunit

MacromoleculeName: Putative pertussis-like toxin subunit / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhi (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: 5 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.753811 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DILLTQSPSI LSVSPGERVS FSCRASQSIG TSIHWYQQKP NGSPRLLIQY ASQSISGIPS RFSGSGSGTD FTLTINSVES EDIADYYCQ HTNGWPYTFG WGDHAGNKP

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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: 5 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.15971 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
VKLVESGGGL VKPGGSLKLS CAASGFAFST YDMSWVRQTP EKRLEWVATI SGGGSYTYYP DIVKGRFTIS RDNARNTLYL QMSSLRSED TALYFCVRQY YGSSNYGMDY WGQGTSVTVS

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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 I / Details: blot for 2.0 second before plunging.
DetailsFreshly prepared size-exclusion-chromatography purified complex between Fab fragment of TyTx4 and Typhoid toxin wild-type

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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: 51.5 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:

4rhr

Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 97594
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


2oz4

chain_id: L

4m48

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-7k7i:
Density-fitted Model Structure of Antibody Variable Domains of TyTx4 in Complex with PltB pentamer of Typhoid Toxin

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