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- PDB-8eht: Cryo-EM reconstruction of the CS20 bacterial adhesion pili -

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Basic information

Entry
Database: PDB / ID: 8eht
TitleCryo-EM reconstruction of the CS20 bacterial adhesion pili
ComponentsCS20 fimbria major subunit protein
KeywordsCELL ADHESION / enterotoxigenic / adhesion pili / superelastic
Function / homologyFimbrial-type adhesion domain superfamily / Adhesion domain superfamily / pilus / cell adhesion / CS20 fimbria major subunit protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDoran, M.H. / Bullitt, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R21AI156236-01 United States
CitationJournal: Structure / Year: 2023
Title: Three structural solutions for bacterial adhesion pilus stability and superelasticity.
Authors: Matthew H Doran / Joseph L Baker / Tobias Dahlberg / Magnus Andersson / Esther Bullitt /
Abstract: Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis ...Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis underpinning the biophysical properties of pili originating from enterotoxigenic (ETEC) and uropathogenic bacteria. Using cryo-electron microscopy we solved the structures of three vaccine target pili from ETEC bacteria, CFA/I, CS17, and CS20. Pairing these and previous pilus structures with force spectroscopy and steered molecular dynamics simulations, we find a strong correlation between subunit-subunit interaction energies and the force required for pilus unwinding, irrespective of genetic similarity. Pili integrate three structural solutions for stabilizing their assemblies: layer-to-layer interactions, N-terminal interactions to distant subunits, and extended loop interactions from adjacent subunits. Tuning of these structural solutions alters the biophysical properties of pili and promotes the superelastic behavior that is essential for sustained bacterial attachment.
History
DepositionSep 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 17, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: CS20 fimbria major subunit protein
F: CS20 fimbria major subunit protein
D: CS20 fimbria major subunit protein
E: CS20 fimbria major subunit protein
W: CS20 fimbria major subunit protein
X: CS20 fimbria major subunit protein
B: CS20 fimbria major subunit protein


Theoretical massNumber of molelcules
Total (without water)122,0467
Polymers122,0467
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Filaments formed under cryo-conditions
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CS20 fimbria major subunit protein


Mass: 17435.199 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: csnA / Production host: Escherichia coli (E. coli) / Strain (production host): WS7179A / References: UniProt: Q8VL73

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: CS20 bacterial adhesion pili / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli) / Strain: WS7179A
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: WS7179A-2(pRA101)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA using the Pelco EasiGlow machine / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / 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 defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 53.62 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5236

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Processing

EM software
IDNameVersionCategory
1RELION3.1.1particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.14model fitting
8PHENIX1.18model fitting
10RELION3.1.1initial Euler assignment
11RELION3.1.1final Euler assignment
13RELION3D reconstruction
14PHENIX1.18model refinement
15ISOLDE1.4model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 111.84 ° / Axial rise/subunit: 8.953 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 9590890
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88048 / Symmetry type: HELICAL

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