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- EMDB-43119: The secreted adhesin EtpA of Enterotoxigenic Escherichia coli in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43119
TitleThe secreted adhesin EtpA of Enterotoxigenic Escherichia coli in complex with the mouse mAb 1G05
Map data
Sample
  • Complex: EtpA in complex with the mAb 1G05 Fab
    • Protein or peptide: EtpA
    • Protein or peptide: mouse mAb 1G05 Heavy Chain
    • Protein or peptide: mouse mAb 1G05 Light Chain
  • Ligand: beta-D-glucopyranose
KeywordsAdhesin / glycoprotein / lectin / antibody-antigen complex / CELL ADHESION
Function / homology
Function and homology information


adhesion of symbiont to host cell surface via host membrane carbohydrate / extracellular region
Similarity search - Function
: / Filamentous haemagglutinin FhaB/tRNA nuclease CdiA-like, TPS domain / TPS secretion domain / haemagglutination activity domain / ESPR domain / Extended Signal Peptide of Type V secretion system / Parallel beta-helix repeat / Parallel beta-helix repeats / Pectin lyase fold / Pectin lyase fold/virulence factor
Similarity search - Domain/homology
Biological speciesEscherichia coli ETEC H10407 (bacteria) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsBerndsen ZT / Ward AB
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI126887 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI089894 United States
Department of Veterans Affairs (VA, United States)5I01BX001469-05). United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32AI007172 United States
Consortia for HIV/AIDS Vaccine DevelopmentUM1 AI144462 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI100663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI113867 United States
CitationJournal: PLoS Pathog / Year: 2024
Title: Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.
Authors: Zachary T Berndsen / Marjahan Akhtar / Mahima Thapa / Tim J Vickers / Aaron Schmitz / Jonathan L Torres / Sabyasachi Baboo / Pardeep Kumar / Nazia Khatoon / Alaullah Sheikh / Melissa Hamrick ...Authors: Zachary T Berndsen / Marjahan Akhtar / Mahima Thapa / Tim J Vickers / Aaron Schmitz / Jonathan L Torres / Sabyasachi Baboo / Pardeep Kumar / Nazia Khatoon / Alaullah Sheikh / Melissa Hamrick / Jolene K Diedrich / Salvador Martinez-Bartolome / Patrick T Garrett / John R Yates / Jackson S Turner / Renee M Laird / Frédéric Poly / Chad K Porter / Jeffrey Copps / Ali H Ellebedy / Andrew B Ward / James M Fleckenstein /
Abstract: Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as ...Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.
History
DepositionDec 12, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43119.png

Downloads & links

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Map

FileDownload / File: emd_43119.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 600 pix.
= 435. Å		0.73 Å/pix.
x 600 pix.
= 435. Å		0.73 Å/pix.
x 600 pix.
= 435. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0739
Minimum - Maximum-0.13314639 - 0.31822848
Average (Standard dev.)0.00090785354 (±0.006345382)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 435.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43119_msk_1.map
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Half map: #2

Fileemd_43119_half_map_1.map
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Half map: #1

Fileemd_43119_half_map_2.map
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Sample components

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Entire : EtpA in complex with the mAb 1G05 Fab

EntireName: EtpA in complex with the mAb 1G05 Fab
Components
  • Complex: EtpA in complex with the mAb 1G05 Fab
    • Protein or peptide: EtpA
    • Protein or peptide: mouse mAb 1G05 Heavy Chain
    • Protein or peptide: mouse mAb 1G05 Light Chain
  • Ligand: beta-D-glucopyranose

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Supramolecule #1: EtpA in complex with the mAb 1G05 Fab

SupramoleculeName: EtpA in complex with the mAb 1G05 Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli ETEC H10407 (bacteria)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: EtpA

MacromoleculeName: EtpA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli ETEC H10407 (bacteria)
Molecular weightTheoretical: 157.456922 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNRIYKLKFD KRRNELVVVS EITTGVGNAK ATGSVEGEKS PRRGVRAMAL SLLSGMMIMA HPAMSANLPT GGQIVAGSGS IQTPSGNQM NIHQNSQNMV ANWNSFDIGK GNTVQFDQPS SSAVALNRVV GGGESQIMGN LKANGQVFLV NPNGVLFGEG A SVSTSGFV ...String:
MNRIYKLKFD KRRNELVVVS EITTGVGNAK ATGSVEGEKS PRRGVRAMAL SLLSGMMIMA HPAMSANLPT GGQIVAGSGS IQTPSGNQM NIHQNSQNMV ANWNSFDIGK GNTVQFDQPS SSAVALNRVV GGGESQIMGN LKANGQVFLV NPNGVLFGEG A SVSTSGFV ASTRDIKNDD FMNRRYTFSG GQKAGAAIVN QGELTTNAGG YIVLAADRVS NSGTIRTPGG KTVLAASERI TL QLDNGGL MSVQVTGDVV NALVENRGLV SARDGQVYLT ALGRGMLMNT VLNVSGVVEA SGMHRQDGNI VLDGGDSGVV HLS GTLQAD NASGQGGKVV VQGKNILLDK GSNITATGGQ GGGEVYVGGG WQGKDSNIRN ADKVVMQGGA RIDVSATQQG NGGT AVLWS DSYTNFHGQI GAKGGETGGN GGRVETSSHG NLQAFGTVSA SAKKGKAGNW LLDSADITIV NGSNVSKTET TQSPP HTQF APTAAGSAVS NTSINNRLNN GTSVTILTHR TRTGTAQGGN ITVNAAINKG NGSDVNLTLQ AGGNITVNNS ITSTEG KLN VNLSGARTSN GSITISNNAN ITTNGGDITV GTTNTSNRVN ISINNTTLNA SNGNIQLTGT GTDSGILFAG NNRLTAS NI ALTGNSTSGN AINLTGTATL NATNNITLTG SSTSGNAINL KGNNTLTASN ITLTGESTSG NAINHTDTTG TTTLNATN N ITMQGTRVQI KHSNITAGNF ALNATVAGSE ISNTTLTATN NINLAAKTNS ASSGVYLKDA RITSTNGSIT ANGTATANG KATHLDGNVT LNASNGRIKL TGNGHGSASG ILFAGNNRLT ASNIALTGNS TSGNAINLTG TATLNATNDI TLTGSSTSGN AINLTGTAT LNATNNITLT GSSTSGNAIN LKGNNTLTAS NITLTGESTS GNAINLTDTT GTTTLNATNN ITMQGTRVQI K HSNITAGN FALNATVAGS EISNTTLTAT NNINLAAKTN SASSGVYLKD ARITSTNGSI TANGTATANG KATHLDGNVT LN ASNGRIK LTGNGHGSAS GILFAGNNRL TASNIALTGN STSGNAINLT GTATLNATND ITLTGSNTSG NAINLTGTAT LNA TNNITL TGSSTSGNAI NLKGNNTLTA SNITLTGEST SGNAINLTDT TGTTTLNATN NITMQGTRVQ IKHSNITAGN FALN ATVAG SEISNTTLTA TNNINLAAKT NSASSGVYLK DARITSTNGS ITANGTATAN GKATHLDGNV TLNASNGRIK LTGNG HGSA SGILFAGNNR LTASNIALTG NSTSGNAINL TGTATLNATN DITLTGSSTS GNAINLTGTA TLNATNNITL TGSSTS GNA INLKGNNTLT ASNITLTGES TSGNAINLTD TTGTTTLNAT NNITMQGTRV QIKHSNITAG NFALNATVAG SEISNTT LT ATNNINLAAK TNSASSGVYL KDARITSTNG SITANGTAPA NDNATYLDGN VTLNASNGSI KLTGNGNGST SGILFAGN N TLTASNITLT GNSEVYWQKL GPEQKLISEE DLNSAVDHHH HHH

UniProtKB: EtpA

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Macromolecule #2: mouse mAb 1G05 Heavy Chain

MacromoleculeName: mouse mAb 1G05 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 51.428785 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSE VQLQQSGAEL VRPGSSVKLS CKASGYIFTN YWIHWVRQRP IQGLEWIGNI DPSESETHYN QNFKDKATL TVDKSSSTVY MHLSSLTSED SSVYYCARGT AVVDFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT A ALGCLVKD ...String:
MGWSCIILFL VATATGVHSE VQLQQSGAEL VRPGSSVKLS CKASGYIFTN YWIHWVRQRP IQGLEWIGNI DPSESETHYN QNFKDKATL TVDKSSSTVY MHLSSLTSED SSVYYCARGT AVVDFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT A ALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SC DKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STY RVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVE WESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

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Macromolecule #3: mouse mAb 1G05 Light Chain

MacromoleculeName: mouse mAb 1G05 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.413287 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSD IQMTQSPSSL SASLGDRVTI SCSASQGISN YLNWYQQKPD GIVKLLIFYT SSLHSGVPSR FSGSGSGTD YSLTISNLEP EDIATYYCQQ YSNLPWTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY P REAKVQWK ...String:
MGWSCIILFL VATATGVHSD IQMTQSPSSL SASLGDRVTI SCSASQGISN YLNWYQQKPD GIVKLLIFYT SSLHSGVPSR FSGSGSGTD YSLTISNLEP EDIATYYCQQ YSNLPWTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY P REAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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Macromolecule #4: beta-D-glucopyranose

MacromoleculeName: beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 38 / Formula: BGC
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BGC:
beta-D-glucopyranose

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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

Concentration1 mg/mL
BufferpH: 6
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 52107
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8vbb:
The secreted adhesin EtpA of Enterotoxigenic Escherichia coli in complex with the mouse mAb 1G05

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