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- PDB-5vxy: Cryo-EM reconstruction of PAK pilus from Pseudomonas aeruginosa -

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Basic information

Entry
Database: PDB / ID: 5vxy
TitleCryo-EM reconstruction of PAK pilus from Pseudomonas aeruginosa
ComponentsFimbrial protein
KeywordsPROTEIN FIBRIL / Melted helix / type IV pili
Function / homology
Function and homology information


pilus / cell adhesion / membrane
Similarity search - Function
Fimbrial protein pilin / Pilin (bacterial filament) / : / Prokaryotic N-terminal methylation site. / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like
Similarity search - Domain/homology
Biological speciesPseudomonas aeruginosa PAK (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8 Å
AuthorsWang, F. / Osinksi, T. / Orlova, A. / Altindal, T. / Craig, L. / Egelman, E.H.
Funding support United States, Canada, France, 4items
OrganizationGrant numberCountry
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 AgencyANR14CE14001002 France
Fondation pour la Recherche Medicale France
CitationJournal: 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.
History
DepositionMay 24, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Sep 20, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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
Revision 1.3Oct 4, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.5Oct 2, 2019Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Derived calculations / Experimental preparation
Category: em_helical_entity / em_sample_support / pdbx_helical_symmetry
Item: _em_helical_entity.axial_rise_per_subunit
Revision 1.6Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
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


Theoretical massNumber of molelcules
Total (without water)315,44521
Polymers315,44521
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, helical filament was observed by negative staining and Cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area75120 Å2
ΔGint-557 kcal/mol
Surface area99100 Å2
SymmetryHelical 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 °)
DetailsThe full assembly is a helical filament. Only a finite portion of the full filament is represented here.

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Components

#1: Protein ...
Fimbrial protein / 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

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

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Sample preparation

ComponentName: Pseudomonas aeruginosa Type IV pilin filament / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa PAK (bacteria) / Strain: PAK/2pfs
Buffer solutionpH: 9 / Details: 50 mM CHES buffer
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage 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)
Image scansMovie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: dev_2471: / Classification: refinement
EM software
IDNameCategory
1EMAN2particle selection
2EPUimage acquisition
4CTFFIND3CTF correction
7Rosettamodel fitting
8UCSF Chimeramodel fitting
10SPIDERinitial Euler assignment
11SPIDERfinal Euler assignment
13SPIDER3D reconstruction
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 87.3 ° / Axial rise/subunit: 10.5 Å / Axial symmetry: C1
3D reconstructionResolution: 8 Å / Resolution method: OTHER / Num. of particles: 31231 / Details: model-map FSC 0.38 cut-off / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0145234
ELECTRON MICROSCOPYf_angle_d1.35982509
ELECTRON MICROSCOPYf_dihedral_angle_d6.62717556
ELECTRON MICROSCOPYf_chiral_restr0.0633717
ELECTRON MICROSCOPYf_plane_restr0.0066909

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