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- PDB-9qb8: Lymphostatin - Conformation II - pH 8 Hepes -

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Basic information

Entry
Database: PDB / ID: 9qb8
TitleLymphostatin - Conformation II - pH 8 Hepes
ComponentsLymphostatin
KeywordsTOXIN / Virulence Factor Lymphostatin LifA Conformation II
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 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBottcher, B. / Schneider, R. / Griessmann, M. / Ramussen, T.
Funding support Germany, 4items
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
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
DepositionFeb 28, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lymphostatin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)366,4762
Polymers366,4211
Non-polymers551
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 Lymphostatin


Mass: 366421.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O127:H6 (bacteria) / Gene: lifA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9RM48
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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 / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.366 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli O127:H6 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2150 mMsodium chlorideNaCl1
35 mMTECEP1
41 mMmanganese chlorideMnCl21
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 4 K

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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: 1100 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: 9.9 sec. / Electron dose: 70 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 28915
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.6particle selectiontemplate picker
2EPUimage acquisition
4cryoSPARC4.6CTF correctionLive session
7UCSF Chimeramodel fitting
12RELION53D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5108934
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298552 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 9euw

9euw


Accession code: 9euw / Source name: PDB / Type: experimental model

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