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- EMDB-73867: Constituent EM map: local refinement of putative MUC2 binding dom... -

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Basic information

Entry
Database: EMDB / ID: EMD-73867
TitleConstituent EM map: local refinement of putative MUC2 binding domain of rEatAp and Fv domain of Fab G12
Map datalocal refinement 1; sharpened map (resampled onto consensus map)
Sample
  • Complex: Binary complex of the recombinant EatA passenger domain with Fab G12
    • Complex: Enterotoxigenic Escherichia coli autotransporter A (EatA) passenger domain
    • Complex: Fragment antigen binding domain of monoclonal antibody G12
KeywordsPROTEASE / BETA-HELIX / SECRETED / MONOCLONAL ANTIBODY / HYDROLASE / HYDROLASE-IMMUNE SYSTEM complex
Biological speciesEscherichia coli (E. coli) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.68 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 10, 2026-
Map releaseJun 10, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73867.png

Downloads & links

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Map

FileDownload / File: emd_73867.map.gz / Format: CCP4 / Size: 172.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal refinement 1; sharpened map (resampled onto consensus map)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (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: 0.225
Minimum - Maximum-1.1836587 - 1.9957097
Average (Standard dev.)-0.00008048816 (±0.013400617)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: local refinement 1; half map A (resampled onto consensus map)

Fileemd_73867_half_map_1.map
Annotationlocal refinement 1; half map A (resampled onto consensus map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: local refinement 1; half map B (resampled onto consensus map)

Fileemd_73867_half_map_2.map
Annotationlocal refinement 1; half map B (resampled onto consensus map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
    • Complex: 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: #1-#3
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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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) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / 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)
FSC plot (resolution estimation)

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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

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