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Yorodumi- PDB-8ptu: 2.5 A cryo-EM structure of the in vitro FimD-catalyzed assembly o... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8ptu | |||||||||||||||
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Title | 2.5 A cryo-EM structure of the in vitro FimD-catalyzed assembly of type 1 pilus rod | |||||||||||||||
Components | Type-1 fimbrial protein, A chain | |||||||||||||||
Keywords | STRUCTURAL PROTEIN / FimA / pilus / monomer / subunit / pili / main structural subunit / high resolution / cryo-EM / helical processing / RELION / Chaperone-usher pilus | |||||||||||||||
Function / homology | Function and homology information cell adhesion involved in single-species biofilm formation / pilus / cell adhesion / identical protein binding Similarity search - Function | |||||||||||||||
Biological species | Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.52 Å | |||||||||||||||
Authors | Zyla, D. / Hospenthal, M. / Glockshuber, R. / Waksman, G. | |||||||||||||||
Funding support | Switzerland, United Kingdom, 4items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ptu.cif.gz | 207 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8ptu.ent.gz | 133.3 KB | Display | PDB format |
PDBx/mmJSON format | 8ptu.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ptu_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8ptu_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8ptu_validation.xml.gz | 33.8 KB | Display | |
Data in CIF | 8ptu_validation.cif.gz | 49.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pt/8ptu ftp://data.pdbj.org/pub/pdb/validation_reports/pt/8ptu | HTTPS FTP |
-Related structure data
Related structure data | 17878MC 6y7sC 8psvC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 / Plasmid: pET11a-FimA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 DE3 / References: UniProt: P04128 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Type 1 pilus rod / Type: COMPLEX Details: Type 1 pilus rod assembled from FimA-FimC complexes in vitro in the presence of the usher FimD activated by FimG/FimF (250 molar excess of FimA-FimC over FimD) Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 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 solution | pH: 7 / Details: in ddH2O. |
Specimen | Conc.: 1.58 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: 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 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 47393 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 7 sec. / Electron dose: 49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 725 |
Image scans | Movie frames/image: 35 / Used frames/image: 1-35 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 115 ° / Axial rise/subunit: 7.8 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 416000 Details: Autopicking based on the 2D classes from manually chosen filaments | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135000 / Algorithm: BACK PROJECTION / Details: standard Relion protocol / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 46.49 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5OH0 Pdb chain-ID: D / Accession code: 5OH0 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 27.04 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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