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- EMDB-73870: Cryo-EM structure of Enterotoxigenic Escherichia coli autotranspo... -

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Basic information

Entry
Database: EMDB / ID: EMD-73870
TitleCryo-EM structure of Enterotoxigenic Escherichia coli autotransporter A (EatA) complexed with the fragment antigen binding domain of monoclonal antibody G12
Map datacomposite map
Sample
  • Complex: Binary complex of the recombinant EatA passenger domain with Fab G12
    • Complex: Enterotoxigenic Escherichia coli autotransporter A (EatA) passenger domain
      • Protein or peptide: Serine protease EatA
    • Complex: Fragment antigen binding domain of monoclonal antibody G12
      • Protein or peptide: Heavy chain of the fragment antigen binding domain of monoclonal antibody G12
      • Protein or peptide: Light chain of the fragment antigen binding domain of monoclonal antibody G12
KeywordsPROTEASE / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBuckley DP / Berndsen ZT
Funding support United States, 4 items
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, 2025-
Header (metadata) releaseJun 3, 2026-
Map releaseJun 3, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73870.png

Downloads & links

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Map

FileDownload / File: emd_73870.map.gz / Format: CCP4 / Size: 172.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 356 pix.
= 306.16 Å		0.86 Å/pix.
x 356 pix.
= 306.16 Å		0.86 Å/pix.
x 356 pix.
= 306.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 11.0
Minimum - Maximum-54.329230000000003 - 82.681849999999997
Average (Standard dev.)0.0030620336 (±1.1973555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions356356356
Spacing356356356
CellA=B=C: 306.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Binary complex of the recombinant EatA passenger domain with Fab G12

EntireName: Binary complex of the recombinant EatA passenger domain with Fab G12
Components
  • Complex: Binary complex of the recombinant EatA passenger domain with Fab G12
    • Complex: Enterotoxigenic Escherichia coli autotransporter A (EatA) passenger domain
      • Protein or peptide: Serine protease EatA
    • Complex: Fragment antigen binding domain of monoclonal antibody G12
      • Protein or peptide: Heavy chain of the fragment antigen binding domain of monoclonal antibody G12
      • Protein or peptide: Light chain of the fragment antigen binding domain of monoclonal antibody G12

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Supramolecule #1: Binary complex of the recombinant EatA passenger domain with Fab G12

SupramoleculeName: Binary complex of the recombinant EatA passenger domain with Fab G12
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: H10407
Molecular weightTheoretical: 23 KDa

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Supramolecule #2: Enterotoxigenic Escherichia coli autotransporter A (EatA) passeng...

SupramoleculeName: Enterotoxigenic Escherichia coli autotransporter A (EatA) passenger domain
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Mature, secreted EatA passenger domain (cleaved N-terminal signal peptide and C-terminal beta-barrel regions) from recombinant expression of full-length EatA
Source (natural)Organism: Escherichia coli (E. coli) / Strain: H10407

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Supramolecule #3: Fragment antigen binding domain of monoclonal antibody G12

SupramoleculeName: Fragment antigen binding domain of monoclonal antibody G12
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Fab fragment generated from papain-digested monoclonal G12 IgG
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Serine protease EatA

MacromoleculeName: Serine protease EatA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
Source (natural)Organism: Escherichia coli (E. coli) / Strain: H10407
Molecular weightTheoretical: 111.014391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ATVNADISYQ TFRDFAENKG AFIVGASNIN IYDKNGVLVG VLDKAPMPDF SSATMNTGTL PPGDHTLYSP QYVVTAKHVN GSDIMSFGH IQNNYTVVGE NNHNSLDIKI RRLNKIVTEV APAEISSVGA VNGAYQEGGR FKAFYRLGGG LQYIKDKNGN L TPVYTNGG ...String:
ATVNADISYQ TFRDFAENKG AFIVGASNIN IYDKNGVLVG VLDKAPMPDF SSATMNTGTL PPGDHTLYSP QYVVTAKHVN GSDIMSFGH IQNNYTVVGE NNHNSLDIKI RRLNKIVTEV APAEISSVGA VNGAYQEGGR FKAFYRLGGG LQYIKDKNGN L TPVYTNGG FLTGGTISAL SSYNNGQMIT APTGDIFNPA NGPLANYLNK GDSGSPLFAY DSLDKKWVLV GVLSSGSEHG NN WVVTTQD FLHQQPKHDF DKTISYDSEK GSLQWRYNKN SGVGTLSQES VVWDMHGKKG GDLNAGKNLQ FTGNNGEIIL HDS IDQGAG YLQFFDNYTV TSLTDQTWTG GGIITEKGVN VLWQVNGVND DNLHKVGEGT LTVNGKGVNN GGLKVGDGTV ILNQ RPDDN GHKQAFSSIN ISSGRATVIL SDANQVNPDK ISWGYRGGTL DLNGNNVNFT RLQAADYGAI VSNNNKNKSE LTLKL QTLN ENDISVDVKT YEVFGGHGSP GDLYYVPASN TYFILKSKAY GPFFSDLDNT NVWQNVGHDR DKAIQIVKQQ KIGESS QPY MFHGQLNGYM DVNIHPLSGK DVLTLDGSVN LPEGVITKKS GTLIFQGHPV IHAGMTTSAG QSDWENRQFT MDKLRLD AA TFHLSRNAHM QGDISAANGS TVILGSSRVF TDKNDGTGNA VSSVEGSSIA TTAGDQSYYS GNVLLENHSS LEVRENFT G GIEAYDSSVS VTSQNAIFDH VGSFVNSSLL LEKGAKLTAQ SGIFTNNTMK IKENASLTLT GIPSVGKPGY YSPVTSTTE GIHLGERASL SVKNMGYLSS NITAENSAAI INLGDSNATI GKTDSPLFST LMRGYNAVLQ GNIMGPQSSV NMNNALWHSD RNSELKELK ANDSQIELGV RGHFAKLRVK ELIASNSVFL VHANNSQADQ LNVTDKLQGS NNTILVDFFN KAANGTNVTL I TAPKGSDE NTFKAGTQQI GFSNITPEIR TENTDTATQW VLTGYQSVAD ARASKIATDF MDSGYKSFLT EVN

UniProtKB: Serine protease EatA

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Macromolecule #2: Heavy chain of the fragment antigen binding domain of monoclonal ...

MacromoleculeName: Heavy chain of the fragment antigen binding domain of monoclonal antibody G12
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.014945 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVESGGG LIQSGGSLRL SCAASGFTFS RYWMHWVRQI PGKGLEWVSR VKGDGTNTDY ADYADSVKGR FTISRDNVKN TMFLQMNSL RVEDTALYHC ARGGYVDNFG KDYNWFDPWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS ...String:
EVQLVESGGG LIQSGGSLRL SCAASGFTFS RYWMHWVRQI PGKGLEWVSR VKGDGTNTDY ADYADSVKGR FTISRDNVKN TMFLQMNSL RVEDTALYHC ARGGYVDNFG KDYNWFDPWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCD

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Macromolecule #3: Light chain of the fragment antigen binding domain of monoclonal ...

MacromoleculeName: Light chain of the fragment antigen binding domain of monoclonal antibody G12
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.983326 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SVTSYELTQP PSASVSPGQT ARITCSGDAL RKQYVYWYQQ RPGQAPVMVM YKDSDRPSGV PERFSGSSSG TTVTLTISGV QAEDEADYY CQSSDSSGSY VIFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP V KAGVETTT ...String:
SVTSYELTQP PSASVSPGQT ARITCSGDAL RKQYVYWYQQ RPGQAPVMVM YKDSDRPSGV PERFSGSSSG TTVTLTISGV QAEDEADYY CQSSDSSGSY VIFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP V KAGVETTT PSKQSNNKYA ASSYLSLTPE QWKSHRSYSC QVTHEGSTVE KTVAPTE

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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

Concentration0.7 mg/mL
BufferpH: 7.4 / Component - Concentration: 1.0 X / Component - Formula: TBS / Component - Name: Tris-Buffered Saline
Details: 10X TBS: 250mM Tris, 27mM potassium chloride, 1.37M sodium chloride, pH 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Added 1X Lauryl Maltose Neopentyl Glycol (LMNG) detergent to sample prior to vitrification..
DetailsThis sample displayed heterogeneity in the ice, resulting in preferred orientation in the final map.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 87.15 K / Max: 87.15 K
SoftwareName: SerialEM (ver. 4)
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number grids imaged: 2 / Number real images: 6382 / Average exposure time: 2.1 sec. / Average electron dose: 49.0 e/Å2
Details: Movies collected during first session: 1972 Movies collected during second session: 4410
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1244157
Details: Initial particle set was obtained by CryoSegNet AI picking, followed by cryoSPARC 2D classification to redo particle picking with 2D templates
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: AB-INITIO
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: cryoSPARC (ver. 4.7.1) / Details: Estimated resolution of composite map. / Number images used: 73337
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: UCSF ChimeraX (ver. 1.8)
DetailsRigid body fitting of AlphaFold-derived models was done in ChimeraX, followed by multiple rounds of local fitting in Coot and Phenix real-space refinement
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-9z77:
Cryo-EM structure of Enterotoxigenic Escherichia coli autotransporter A (EatA) complexed with the fragment antigen binding domain of monoclonal antibody G12

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