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- PDB-9euv: Lymphostatin, conformation 1 (composite structure) -

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Basic information

Entry
Database: PDB / ID: 9euv
TitleLymphostatin, conformation 1 (composite structure)
ComponentsEfa1/LifA protein
KeywordsTOXIN / bacterial virulence factor / glycosyltransferase / protease
Function / homology
Function and homology information


glycosyltransferase activity / cysteine-type endopeptidase activity
Similarity search - Function
Protein of unknown function DUF3491 / Protein of unknown function (DUF3491) / Peptidase C58, YopT-type domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesEscherichia coli O127:H6 str. E2348/69 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsGriessmann, M. / Rasmussen, T. / Bottcher, B.
Funding support Germany, 8items
OrganizationGrant numberCountry
German Research Foundation (DFG)359471283 Germany
German Research Foundation (DFG)456578072 Germany
German Research Foundation (DFG)525040890 Germany
German Research Foundation (DFG)428774170 Germany
German Research Foundation (DFG)INST 93/903-1 Germany
German Research Foundation (DFG)INST 93/1042-1 Germany
German Research Foundation (DFG)INST 93/1143-1 Germany
German Research Foundation (DFG)BO1150/18-1 Germany
CitationJournal: Nat Commun / Year: 2025
Title: Structure of lymphostatin, a large multi-functional virulence factor of pathogenic Escherichia coli.
Authors: Matthias Griessmann / Tim Rasmussen / Vanessa J Flegler / Christian Kraft / Ronja Schneider / Max Hateley / Lukas Spantzel / Mark P Stevens / Michael Börsch / Bettina Böttcher /
Abstract: Lymphostatin is a key virulence factor of enteropathogenic and enterohaemorrhagic Escherichia coli, playing roles in bacterial colonisation of the gut and in the inhibition of lymphocyte ...Lymphostatin is a key virulence factor of enteropathogenic and enterohaemorrhagic Escherichia coli, playing roles in bacterial colonisation of the gut and in the inhibition of lymphocyte proliferation and proinflammatory responses. The protein's glycosyltransferase and cysteine protease motifs are required for activity against lymphocytes, but high-resolution structural information has proven elusive. Here, we describe the structure of lymphostatin from enteropathogenic E. coli O127:H6, determined by electron cryo-microscopy at different pH values. We observe three conformations of a highly complex molecule with two glycosyltransferase domains, one PaToxP-like protease domain, an ADP-ribosyltransferase domain, a vertex domain and a delivery domain. Long linkers hold these domains together and occlude the catalytic sites of the N-terminal glycosyltransferase and protease domains. Lymphostatin binds to bovine T-lymphocytes and HEK-293T cells, forming clusters at the plasma membrane that are internalized. With six distinct domains, lymphostatin can be regarded as a multitool of pathogenic Escherichia coli, enabling complex interactions with host cells.
History
DepositionMar 28, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 9, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Jul 23, 2025Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Efa1/LifA protein


Theoretical massNumber of molelcules
Total (without water)367,2341
Polymers367,2341
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 Efa1/LifA protein / Lymphostatin


Mass: 367234.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal His6 tag
Source: (gene. exp.) Escherichia coli O127:H6 str. E2348/69 (bacteria)
Gene: E2348C_3234 / Production host: Escherichia coli (E. coli) / Strain (production host): DH5a / References: UniProt: B7UI23
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

ComponentName: Lymphostatin / Type: COMPLEX / Details: C-terminal His6 tag / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli O127:H6 str. E2348/69 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: DH5a
Buffer solutionpH: 4
Details: pH was adjusted before vitrification to 4.0 with hydrochloric acid
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMsodium dihydrogen phosphateNaH2PO41
2100 mMsodium chlorideNaCl1
30.075 mMmanganese chlorideMnCl21
41.5 mMTCEPC9H15O6PHCl1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 1400 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.1 sec. / Electron dose: 70 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 41392
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2EPU3.5image acquisition
4cryoSPARC4.4CTF correction
7Coot0.9.8model fitting
9cryoSPARCinitial Euler assignment
10cryoSPARC4.4final Euler assignment
12RELION53D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13373262
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120944
Details: This is a composite map from two original maps. One overall map was used until residue 2199 and for overall alignment. The second map was a local refinement for the C-terminal part from ...Details: This is a composite map from two original maps. One overall map was used until residue 2199 and for overall alignment. The second map was a local refinement for the C-terminal part from residue 2200. Phenix Combine focused maps tool was used (version 1.20.1)
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingDetails: modelangelo was used for the initial model. / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00222723
ELECTRON MICROSCOPYf_angle_d0.42930789
ELECTRON MICROSCOPYf_dihedral_angle_d3.4453044
ELECTRON MICROSCOPYf_chiral_restr0.0393490
ELECTRON MICROSCOPYf_plane_restr0.0043970

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