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- PDB-8psv: 2.7 A cryo-EM structure of in vitro assembled type 1 pilus rod -

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Basic information

Entry
Database: PDB / ID: 8psv
Title2.7 A cryo-EM structure of in vitro assembled type 1 pilus rod
ComponentsType-1 fimbrial protein, A chain
KeywordsSTRUCTURAL PROTEIN / FimA / pilus / monomer / subunit / pili / main structural subunit / high resolution / cryo-EM / helical processing / RELION / Chaperone-usher pilus
Function / homologycell adhesion involved in single-species biofilm formation / Fimbrial-type adhesion domain / Fimbrial protein / Fimbrial-type adhesion domain superfamily / Adhesion domain superfamily / pilus / cell adhesion / identical protein binding / Type-1 fimbrial protein, A chain
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHospenthal, M. / Zyla, D. / Glockshuber, R. / Waksman, G.
Funding support Switzerland, United Kingdom, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_176403/1 Switzerland
Swiss National Science Foundation31003A_156304 Switzerland
Swiss National Science Foundation310030_201234 Switzerland
Medical Research Council (MRC, United Kingdom)018434 United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.
Authors: Dawid S Zyla / Thomas Wiegand / Paul Bachmann / Rafal Zdanowicz / Christoph Giese / Beat H Meier / Gabriel Waksman / Manuela K Hospenthal / Rudi Glockshuber /
Abstract: Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of ...Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.
History
DepositionJul 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2024Group: 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: Type-1 fimbrial protein, A chain
B: Type-1 fimbrial protein, A chain
C: Type-1 fimbrial protein, A chain
D: Type-1 fimbrial protein, A chain
E: Type-1 fimbrial protein, A chain
F: Type-1 fimbrial protein, A chain


Theoretical massNumber of molelcules
Total (without water)108,7266
Polymers108,7266
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Type-1 fimbrial protein, A chain / Type-1A pilin


Mass: 18121.074 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: fimA, pilA, b4314, JW4277 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04128

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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: Type 1 pilus rod / Type: COMPLEX
Details: Type 1 pili assembled in vitro from recombinantly expressed as inclusion bodies and refolded FimA.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 20.3 kDa/nm / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli str. K-12 substr. W3110 (bacteria)
Buffer solutionpH: 7 / Details: in ddH2O.
SpecimenConc.: 1.58 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 277 K
Details: 3 ul sample, 30 s wait time, 0.5 s drain time, 6 s blotting

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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: 94340 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 14 sec. / Electron dose: 97.54 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansWidth: 8000 / Height: 8000 / Movie frames/image: 56

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9RELION3.08initial Euler assignment
10RELION3.08final Euler assignment
11RELION3.08classification
12RELION3.083D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 115 ° / Axial rise/subunit: 7.76 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 516000
Details: Autopicking based on the 2D classes from manually chosen filaments
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11000 / Symmetry type: HELICAL
Atomic model buildingB value: 45.07 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5OH0

5oh0


Pdb chain-ID: D / Accession code: 5OH0 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 23.88 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00266714
ELECTRON MICROSCOPYf_angle_d0.39869174
ELECTRON MICROSCOPYf_chiral_restr0.03921146
ELECTRON MICROSCOPYf_plane_restr0.00231230
ELECTRON MICROSCOPYf_dihedral_angle_d4.0091972

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