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- PDB-8cio: Cryo-EM structure of the CupE pilus from Pseudomonas aeruginosa -

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Basic information

Entry
Database: PDB / ID: 8cio
TitleCryo-EM structure of the CupE pilus from Pseudomonas aeruginosa
ComponentsSCPU domain-containing protein
KeywordsPROTEIN FIBRIL / Pseudomonas aeruginosa / pilus / Chaperone-Usher / Chaperone-Usher Pathway / adhesin / biofilm
Function / homologySpore coat protein U / Spore Coat Protein U domain / Spore Coat Protein U domain / Spore coat protein U domain-containing protein
Function and homology information
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBoehning, J. / Bharat, T.A.M.
Funding support United Kingdom, United States, 7items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Royal Society202231/Z/16/Z United Kingdom
The Vallee Foundation Inc. United States
Leverhulme Trust United Kingdom
The Lister Institute of Preventive Medicine United Kingdom
Medical Research Council (MRC, United Kingdom)MR/K501256/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N013468/1 United Kingdom
CitationJournal: PLoS Pathog / Year: 2023
Title: Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa.
Authors: Jan Böhning / Adrian W Dobbelstein / Nina Sulkowski / Kira Eilers / Andriko von Kügelgen / Abul K Tarafder / Sew-Yeu Peak-Chew / Mark Skehel / Vikram Alva / Alain Filloux / Tanmay A M Bharat /
Abstract: Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively ...Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE1 subunits within the pilus are arranged in a zigzag architecture, containing an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, with comparatively weaker interactions at the rest of the inter-subunit interface. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which might facilitate their role in promoting cellular attachment. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili, providing a structural basis for understanding their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.
History
DepositionFeb 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SCPU domain-containing protein
B: SCPU domain-containing protein
C: SCPU domain-containing protein
D: SCPU domain-containing protein
E: SCPU domain-containing protein


Theoretical massNumber of molelcules
Total (without water)80,6395
Polymers80,6395
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
SCPU domain-containing protein


Mass: 16127.815 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa PAO1 (bacteria) / References: UniProt: Q9HVE4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: CupE pilus / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: PAO1 mutant / Cellular location: Cell surface
Buffer solutionpH: 7.4 / Details: Phosphate-buffered saline
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2image acquisition
4CTFFIND4CTF correction
7Coot0.9model fitting
12RELION3.13D reconstruction
13PHENIX1.2model refinement
CTF correctionDetails: As implemented in RELION 3.1 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -145.55 ° / Axial rise/subunit: 33.04 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 3766858
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 274457 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Initial local fitting of a homology model was done using ChimeraX, and the structure manually re-built in Coot.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025740
ELECTRON MICROSCOPYf_angle_d0.5347890
ELECTRON MICROSCOPYf_dihedral_angle_d3.641835
ELECTRON MICROSCOPYf_chiral_restr0.0481005
ELECTRON MICROSCOPYf_plane_restr0.0031015

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