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- PDB-9z78: Cryo-EM structure of Enterotoxigenic Escherichia coli autotranspo... -

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Basic information

Entry
Database: PDB / ID: 9z78
TitleCryo-EM structure of Enterotoxigenic Escherichia coli autotransporter A (EatA) complexed with the fragment antigen binding domain of monoclonal antibody 15
Components
  • Heavy chain of the fragment antigen binding domain of monoclonal antibody 15
  • Light chain of the fragment antigen binding domain of monoclonal antibody 15
  • Serine protease EatA
KeywordsHYDROLASE/IMMUNE SYSTEM / PROTEASE / BETA-HELIX / SECRETED / MONOCLONAL ANTIBODY / HYDROLASE / HYDROLASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / cell outer membrane / periplasmic space / serine-type endopeptidase activity / cell surface / proteolysis / extracellular region
Similarity search - Function
: / PIC/HAP1/IgA0 second beta-solenoid repeat region / : / Peptidase S6, IgA endopeptidase / Peptidase family S6 domain / Immunoglobulin A1 protease / Peptidase family S6 domain profile. / Autotransporter beta-domain / Outer membrane autotransporter barrel / Autotransporter beta-domain ...: / PIC/HAP1/IgA0 second beta-solenoid repeat region / : / Peptidase S6, IgA endopeptidase / Peptidase family S6 domain / Immunoglobulin A1 protease / Peptidase family S6 domain profile. / Autotransporter beta-domain / Outer membrane autotransporter barrel / Autotransporter beta-domain / Autotransporter beta-domain profile. / Autotransporter beta-domain / Autotransporter beta-domain superfamily / Autotransporter, pectate lyase C-like domain superfamily / Pectin lyase fold/virulence factor / Peptidase S1, PA clan
Similarity search - Domain/homology
Serine protease EatA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsBuckley, D.P. / Berndsen, Z.T.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI089894 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI126887 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32AI007172 United States
Department of Veterans Affairs (VA, United States)5I01BX001469-05 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Human enterotoxigenic (ETEC) infections elicit antibodies that broadly neutralize mucinases of pathogenic and .
Authors: David P Buckley / Marjahan Akhtar / Mahima Thapa / Aaron Schmitz / Jackson Turner / Tim J Vickers / Nazia Khatoon / M Hasanul Kaisar / Jonathan A Coggin / Debayan Ganguli / Alaullah Sheikh / ...Authors: David P Buckley / Marjahan Akhtar / Mahima Thapa / Aaron Schmitz / Jackson Turner / Tim J Vickers / Nazia Khatoon / M Hasanul Kaisar / Jonathan A Coggin / Debayan Ganguli / Alaullah Sheikh / Renee M Laird / Frédéric Poly / Chad K Porter / Fernando Ruiz-Perez / Mark J Miller / Fahima Chowdhury / Tafiqur R Bhuiyan / Firdausi Qadri / Sergio Trillo-Muyo / Brendan Dolan / Sjoerd van der Post / Ali Ellebedy / Zachary T Berndsen / James M Fleckenstein /
Abstract: Enterotoxigenic (ETEC) and are the most common bacterial diarrheal pathogens among young children of low-middle income regions. Enteric pathogens must overcome formidable host defenses, including ...Enterotoxigenic (ETEC) and are the most common bacterial diarrheal pathogens among young children of low-middle income regions. Enteric pathogens must overcome formidable host defenses, including the protective barrier formed by intestinal mucus. ETEC produce a virulence protein called EatA, a member of the Serine Protease Autotransporter of the (SPATE) family, where the secreted passenger domain (EatA) specifically degrades MUC2, the major mucus secreted by goblet cells of the human intestine. Notably, some spp., as well as other diarrheagenic pathovars, secrete homologues of EatA known as SepA, and Pic. Here, we demonstrate that EatA, SepA, and Pic are functionally redundant MUC2 mucinases and that recombinant monoclonal antibodies (mAbs) derived from plasmablasts of ETEC-infected humans can inhibit MUC2 degradation by all three proteases. We present cryo-EM structures of EatA and the related SPATE proteins, SepA, and Pic, complexed to fragment antigen-binding portions of these mAbs to demonstrate that those targeting a core β-helix epitope shared by all three SPATE molecules broadly neutralize the capacity to degrade MUC2. These mAbs effectively prevent MUC2 degradation by each SPATE as well as mucus penetration by ETEC, , and Pic-producing enteroaggregative (EAEC). We anticipate that these studies could facilitate rational design of vaccines that broadly protect against major enteric pathogens by targeting a shared virulence feature.
History
DepositionNov 16, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 1, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine protease EatA
B: Heavy chain of the fragment antigen binding domain of monoclonal antibody 15
C: Light chain of the fragment antigen binding domain of monoclonal antibody 15


Theoretical massNumber of molelcules
Total (without water)158,3773
Polymers158,3773
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Serine protease EatA / Autotransporter protein EatA / ETEC autotransporter A


Mass: 111014.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: H10407 / Gene: eatA, ETEC_p948_0020 / Plasmid: pTW5016
Details (production host): eatA cloned into BamHI/SalI sites on pWSK29
Production host: Escherichia coli (E. coli) / Strain (production host): JF5516
References: UniProt: Q84GK0, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#2: Antibody Heavy chain of the fragment antigen binding domain of monoclonal antibody 15


Mass: 23874.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#3: Antibody Light chain of the fragment antigen binding domain of monoclonal antibody 15


Mass: 23488.154 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
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
IDNameTypeEntity IDParent-IDSourceDetails (eV)
1Binary complex of the recombinant EatA passenger domain with Fab 15COMPLEXall0RECOMBINANT
2Enterotoxigenic Escherichia coli autotransporter A (EatA) passenger domainCOMPLEX#11RECOMBINANTMature, secreted EatA passenger domain (cleaved N-terminal signal peptide and C-terminal beta-barrel regions) from recombinant expression of full-length EatA
3Fragment antigen binding domain of monoclonal antibody 15COMPLEX#2-#31RECOMBINANTFab fragment generated from papain-digested monoclonal 15 IgG
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.158 MDaNO
210.111 MDaNO
310.047 MDaNO
410.023 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)562H10407
32Escherichia coli (E. coli)562H10407
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCell
21Escherichia coli (E. coli)562JF5516
32Escherichia coli (E. coli)562JF5516
43Homo sapiens (human)9606HEK293
Buffer solutionpH: 7.4
Details: 10X TBS: 250mM Tris, 27mM potassium chloride, 1.37M sodium chloride, pH 7.4
Buffer componentConc.: 1 X / Name: Tris-Buffered Saline / Formula: TBS
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Added 1X Lauryl Maltose Neopentyl Glycol (LMNG) detergent to sample prior to vitrification.

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8.72 sec. / Electron dose: 52 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5394

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
2PHENIX1.21_5207model refinement
5cryoSPARC4.7.1CTF correction
10cryoSPARC4.7.1initial Euler assignment
11cryoSPARC4.7.1final Euler assignment
13cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2336121
Details: Initial particle set was obtained by CryoSegNet AI picking, followed by cryoSPARC 2D classification to redo particle picking with 2D templates
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1981936 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Rigid body fitting of AlphaFold-derived models was done in ChimeraX, followed by multiple rounds of local fitting in Coot and Phenix real-space refinement
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 46.27 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00349580
ELECTRON MICROSCOPYf_angle_d0.675612993
ELECTRON MICROSCOPYf_chiral_restr0.04851471
ELECTRON MICROSCOPYf_plane_restr0.00611688
ELECTRON MICROSCOPYf_dihedral_angle_d5.84491317

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