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- EMDB-73873: Cryo-EM structure of Secreted extracellular protein A (SepA) from... -

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Basic information

Entry
Database: EMDB / ID: EMD-73873
TitleCryo-EM structure of Secreted extracellular protein A (SepA) from Shigella flexneri complexed with the fragment antigen binding domain of monoclonal antibody 40
Map datasharpened map
Sample
  • Complex: Binary complex of the recombinant SepA passenger domain with Fab 40
    • Complex: Secreted extracellular protein A (SepA) from Shigella flexneri passenger domain
      • Protein or peptide: Serine protease SepA autotransporter
    • Complex: Fragment antigen binding domain of monoclonal antibody 40
      • Protein or peptide: Heavy chain of the fragment antigen binding domain of monoclonal antibody 40
      • Protein or peptide: Light chain of the fragment antigen binding domain of monoclonal antibody 40
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 SepA autotransporter
Similarity search - Component
Biological speciesShigella flexneri 2a str. 2457T (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.99 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_73873.png

Downloads & links

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Map

FileDownload / File: emd_73873.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 320 pix.
= 300.16 Å		0.94 Å/pix.
x 320 pix.
= 300.16 Å		0.94 Å/pix.
x 320 pix.
= 300.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.938 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.192
Minimum - Maximum-0.69119 - 1.2965721
Average (Standard dev.)0.0013826335 (±0.020987857)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 300.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_73873_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_73873_half_map_2.map
Annotationhalf map A
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 SepA passenger domain with Fab 40

EntireName: Binary complex of the recombinant SepA passenger domain with Fab 40
Components
  • Complex: Binary complex of the recombinant SepA passenger domain with Fab 40
    • Complex: Secreted extracellular protein A (SepA) from Shigella flexneri passenger domain
      • Protein or peptide: Serine protease SepA autotransporter
    • Complex: Fragment antigen binding domain of monoclonal antibody 40
      • Protein or peptide: Heavy chain of the fragment antigen binding domain of monoclonal antibody 40
      • Protein or peptide: Light chain of the fragment antigen binding domain of monoclonal antibody 40

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Supramolecule #1: Binary complex of the recombinant SepA passenger domain with Fab 40

SupramoleculeName: Binary complex of the recombinant SepA passenger domain with Fab 40
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Shigella flexneri 2a str. 2457T (bacteria)
Molecular weightTheoretical: 23 KDa

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Supramolecule #2: Secreted extracellular protein A (SepA) from Shigella flexneri pa...

SupramoleculeName: Secreted extracellular protein A (SepA) from Shigella flexneri passenger domain
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Mature, secreted SepA passenger domain (cleaved N-terminal signal peptide and C-terminal beta-barrel regions) from recombinant expression of full-length SepA
Source (natural)Organism: Shigella flexneri 2a str. 2457T (bacteria)

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Supramolecule #3: Fragment antigen binding domain of monoclonal antibody 40

SupramoleculeName: Fragment antigen binding domain of monoclonal antibody 40
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Fab fragment generated from papain-digested monoclonal 40 IgG
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Serine protease SepA autotransporter

MacromoleculeName: Serine protease SepA autotransporter / 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: Shigella flexneri 2a str. 2457T (bacteria)
Molecular weightTheoretical: 109.400359 KDa
Recombinant expressionOrganism: Escherichia coli DH5[alpha] (bacteria)
SequenceString: ATVSAEIPYQ IFRDFAENKG QFTPGTTNIS IYDKQGNLVG KLDKAPMADF SSATITTGSL PPGDHTLYSP QYVVTAKHVS GSDTMSFGY AKNTYTAVGT NNNSGLDIKT RRLSKLVTEV APAEVSDIGA VSGAYQAGGR FTEFYRLGGG MQYVKDKNGN R TQVYTNGG ...String:
ATVSAEIPYQ IFRDFAENKG QFTPGTTNIS IYDKQGNLVG KLDKAPMADF SSATITTGSL PPGDHTLYSP QYVVTAKHVS GSDTMSFGY AKNTYTAVGT NNNSGLDIKT RRLSKLVTEV APAEVSDIGA VSGAYQAGGR FTEFYRLGGG MQYVKDKNGN R TQVYTNGG FLVGGTVSAL NSYNNGQMIT AQTGDIFNPA NGPLANYLNM GDSGSPLFAY DSLQKKWVLI GVLSSGTNYG NN WVVTTQD FLGQQPQNDF DKTIAYTSGE GVLQWKYDAA NGTGTLTQGN TTWDMHGKKG NDLNAGKNLL FTGNNGEVVL QNS VNQGAG YLQFAGDYRV SALNGQTWMG GGIITDKGTH VLWQVNGVAG DNLHKTGEGT LTVNGTGVNA GGLKVGDGTV ILNQ QADAD GKVQAFSSVG IASGRPTVVL SDSQQVNPDN ISWGYRGGRL ELNGNNLTFT RLQAADYGAI ITNNSEKKST VTLDL QTLK ASDINVPVNT VSIFGGRGAP GDLYYDSSTK QYFILKASSY SPFFSDLNNS SVWQNVGKDR NKAIDTVKQQ KIEASS QPY MYHGQLNGNM DVNIPQLSGK DVLALDGSVN LPEGSITKKS GTLIFQGHPV IHAGTTTSSS QSDWETRQFT LEKLKLD AA TFHLSRNGKM QGDINATNGS TVILGSSRVF TDRSDGTGNA VSSVEGSATA TTVGDQSDYS GNVTLENKSS LQIMERFT G GIEAYDSTVS VTSQNAVFDR VGSFVNSSLT LGKGAKLTAQ SGIFSTGAVD VKENASLTLT GMPSAQKQGY YSPVISTTE GINLEDNASF SVKNMGYLSS DIHAGTTAAT INLGDSDADA GKTDSPLFSS LMKGYNAVLR GSITGAQSTV NMINALWYSD GKSEAGALK AKGSRIELGD GKHFATLQVK ELSADNTTFL MHTNNSRADQ LNVTDKLSGS NNSVLVDFLN KPASEMSVTL I TAPKGSDE KTFTAGTQQI GFSNVTPVIS TEKTDDATKW VLTGYQTTAD AGASKAAKDF MASGYKSFLT EVNNL

UniProtKB: Serine protease SepA autotransporter

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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 40
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.757502 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLQESGPG LVKPSETLSL TCTVSGASIS SSSYYCIWVR QSPGKGLEWI GTTYYSGTTY HEPSLRSRVS ISLDTSKNQF SLMLTSVTA ADTARYYCAI SYGWGSYVNH WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QVQLQESGPG LVKPSETLSL TCTVSGASIS SSSYYCIWVR QSPGKGLEWI GTTYYSGTTY HEPSLRSRVS ISLDTSKNQF SLMLTSVTA ADTARYYCAI SYGWGSYVNH WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCD

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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 40
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.534123 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EIVLTQSPAT LSLSPGERAT LSCRASQIFG SDLAWYQHKP GQAPRLLIFN TSNRVTGTPA RFSGSGSGTD FTLTINSLEP DDVAVYYCH QRSSWPSITF GQGTRLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
EIVLTQSPAT LSLSPGERAT LSCRASQIFG SDLAWYQHKP GQAPRLLIFN TSNRVTGTPA RFSGSGSGTD FTLTINSLEP DDVAVYYCH QRSSWPSITF GQGTRLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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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 was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 87.15 K / Max: 87.15 K
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
SoftwareName: EPU / Details: TFS EPU
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 9623 / Average exposure time: 4.36 sec. / Average electron dose: 41.0 e/Å2
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: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2865944
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: 1.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 663017
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
Output model

PDB-9z7a:
Cryo-EM structure of Secreted extracellular protein A (SepA) from Shigella flexneri complexed with the fragment antigen binding domain of monoclonal antibody 40

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