National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
AI114902
United States
Canadian Institutes of Health Research (CIHR)
MOP125959
Canada
French National Research Agency
ANR14CE14001002
France
Fondation pour la Recherche Medicale
France
Citation
Journal: Structure / Year: 2017 Title: Cryoelectron Microscopy Reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae Type IV Pili at Sub-nanometer Resolution. Authors: Fengbin Wang / Mathieu Coureuil / Tomasz Osinski / Albina Orlova / Tuba Altindal / Gaël Gesbert / Xavier Nassif / Edward H Egelman / Lisa Craig / Abstract: We report here cryoelectron microscopy reconstructions of type IV pili (T4P) from two important human pathogens, Pseudomonas aeruginosa and Neisseria gonorrhoeae, at ∼ 8 and 5 Å resolution, ...We report here cryoelectron microscopy reconstructions of type IV pili (T4P) from two important human pathogens, Pseudomonas aeruginosa and Neisseria gonorrhoeae, at ∼ 8 and 5 Å resolution, respectively. The two structures reveal distinct arrangements of the pilin globular domains on the pilus surfaces, which impart different helical parameters, but similar packing of the conserved N-terminal α helices, α1, in the filament core. In contrast to the continuous α helix seen in the X-ray crystal structures of the P. aeruginosa and N. gonorrhoeae pilin subunits, α1 in the pilus filaments has a melted segment located between conserved helix-breaking residues Gly14 and Pro22, as seen for the Neisseria meningitidis T4P. Using mutagenesis we show that Pro22 is critical for pilus assembly, as are Thr2 and Glu5, which are positioned to interact in the hydrophobic filament core. These structures provide a framework for understanding T4P assembly, function, and biophysical properties.
A: Fimbrial protein B: Fimbrial protein C: Fimbrial protein D: Fimbrial protein E: Fimbrial protein F: Fimbrial protein G: Fimbrial protein H: Fimbrial protein I: Fimbrial protein J: Fimbrial protein K: Fimbrial protein L: Fimbrial protein M: Fimbrial protein N: Fimbrial protein O: Fimbrial protein P: Fimbrial protein Q: Fimbrial protein R: Fimbrial protein S: Fimbrial protein T: Fimbrial protein U: Fimbrial protein
Evidence: microscopy, helical filament was observed by negative staining and Cryo-EM
Type
Name
Symmetry operation
Number
identity operation
1_555
1
Buried area
75120 Å2
ΔGint
-557 kcal/mol
Surface area
99100 Å2
Symmetry
Helical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 21 / Rise per n subunits: 10.5 Å / Rotation per n subunits: 87.3 °)
Details
The full assembly is a helical filament. Only a finite portion of the full filament is represented here.
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Components
#1: Protein
... Fimbrialprotein / Type IV pilin / Pilin
Mass: 15021.180 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa PAK (bacteria) / Strain: PAK/2pfs / References: UniProt: P02973
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction
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Sample preparation
Component
Name: Pseudomonas aeruginosa Type IV pilin filament / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weight
Experimental value: NO
Source (natural)
Organism: Pseudomonas aeruginosa PAK (bacteria) / Strain: PAK/2pfs
Buffer solution
pH: 9 / Details: 50 mM CHES buffer
Specimen
Conc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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
Image recording
Average exposure time: 2 sec. / Electron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the ...Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the estimation of the CTF as well as for boxing filaments. Only the first two parts were used for the reconstruction (~5 electrons per Angstrom^2)
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