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- PDB-9z7a: Cryo-EM structure of Secreted extracellular protein A (SepA) from... -

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Basic information

Entry
Database: PDB / ID: 9z7a
TitleCryo-EM structure of Secreted extracellular protein A (SepA) from Shigella flexneri complexed with the fragment antigen binding domain of monoclonal antibody 40
Components
  • Heavy chain of the fragment antigen binding domain of monoclonal antibody 40
  • Light chain of the fragment antigen binding domain of monoclonal antibody 40
  • Serine protease SepA autotransporter
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
: / 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 ...: / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.99 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: To Be Published
Title: Human infection with enterotoxigenic E. coli elicits antibodies that broadly neutralize mucin-degrading proteases of pathogenic E. coli and Shigella
Authors: Buckley, D.P. / Akhtar, M. / Thapa, M. / Schmitz, A. / Turner, J. / Vickers, T.J. / Khatoon, N. / Kaisar, H. / Coggin, J.A. / Ganguli, D. / Sheikh, A. / Laird, R.M. / Poly, F. / Porter, C.K. ...Authors: Buckley, D.P. / Akhtar, M. / Thapa, M. / Schmitz, A. / Turner, J. / Vickers, T.J. / Khatoon, N. / Kaisar, H. / Coggin, J.A. / Ganguli, D. / Sheikh, A. / Laird, R.M. / Poly, F. / Porter, C.K. / Ruiz-Perez, F. / Miller, M.J. / Bhuiyan, T.R. / Qadri, F. / Trillo-Muyo, S. / Dolan, B. / van der Post, S. / Ellebedy, A. / Berndsen, Z.T. / Fleckenstein, J.M.
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine protease SepA autotransporter
B: Heavy chain of the fragment antigen binding domain of monoclonal antibody 40
C: Light chain of the fragment antigen binding domain of monoclonal antibody 40


Theoretical massNumber of molelcules
Total (without water)156,6923
Polymers156,6923
Non-polymers00
Water00
1


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

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Components

#1: Protein Serine protease SepA autotransporter


Mass: 109400.359 Da / Num. of mol.: 1
Mutation: F684S natural variant compared to Q8VSL2 UniProt entry (F740 in UniProt)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri 2a str. 2457T (bacteria)
Gene: sepA, CP0070 / Plasmid: pTV006
Details (production host): sepA from Shigella flexneri 2a cloned into pBAD/mycHisB, AmpR
Production host: Escherichia coli DH5[alpha] (bacteria)
References: UniProt: Q8VSL2, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#2: Antibody Heavy chain of the fragment antigen binding domain of monoclonal antibody 40


Mass: 23757.502 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 40


Mass: 23534.123 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 SepA passenger domain with Fab 40COMPLEXall0RECOMBINANT
2Secreted extracellular protein A (SepA) from Shigella flexneri passenger domainCOMPLEX#11RECOMBINANTMature, secreted SepA passenger domain (cleaved N-terminal signal peptide and C-terminal beta-barrel regions) from recombinant expression of full-length SepA
3Fragment antigen binding domain of monoclonal antibody 40COMPLEX#2-#31RECOMBINANTFab fragment generated from papain-digested monoclonal 40 IgG
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.157 MDaNO
210.109 MDaNO
310.047 MDaNO
410.023 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Shigella flexneri 2a str. 2457T (bacteria)198215
32Shigella flexneri 2a str. 2457T (bacteria)198215
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
21Escherichia coli DH5[alpha] (bacteria)668369
32Escherichia coli DH5[alpha] (bacteria)668369
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Temperature (max): 87.15 K / Temperature (min): 87.15 K
Image recordingAverage exposure time: 4.36 sec. / Electron dose: 41 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9623
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7.1particle selection
2EPUimage acquisitionTFS EPU
4cryoSPARC4.7.1CTF correction
7UCSF ChimeraX1.8model fitting
8Coot0.9.8.7model fitting
10PHENIX1.21.2_5419model refinement
11cryoSPARC4.7.1initial Euler assignment
12cryoSPARC4.7.1final Euler assignment
14cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2865944
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: 1.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 663017 / 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: 60.36 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00479178
ELECTRON MICROSCOPYf_angle_d0.729612466
ELECTRON MICROSCOPYf_chiral_restr0.05021428
ELECTRON MICROSCOPYf_plane_restr0.00561617
ELECTRON MICROSCOPYf_dihedral_angle_d5.67251272

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