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- PDB-5vxx: Cryo-EM reconstruction of Neisseria gonorrhoeae Type IV pilus -

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Basic information

Entry
Database: PDB / ID: 5vxx
TitleCryo-EM reconstruction of Neisseria gonorrhoeae Type IV pilus
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
PHOSPHORIC ACID MONO-(2-AMINO-ETHYL) ESTER / Type IV major pilin protein PilE1
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsWang, F. / 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 1.4Oct 3, 2018Group: Data collection / Experimental preparation / Refinement description
Category: em_sample_support / refine
Revision 1.5Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-8739
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Structure viewerMolecule:
MolmilJmol/JSmol

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)372,66563
Polymers361,12621
Non-polymers11,53942
Water0
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 area94500 Å2
ΔGint-280 kcal/mol
Surface area104770 Å2
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 / MS11 antigen / Pilin


Mass: 17196.467 Da / Num. of mol.: 21 / Mutation: P69S, S71T / Source method: isolated from a natural source / Source: (natural) Neisseria gonorrhoeae (bacteria) / Strain: C30 / References: UniProt: P02974
#2: Polysaccharide...
alpha-D-galactopyranose-(1-3)-2,4-bisacetamido-2,4,6-trideoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 408.401 Da / Num. of mol.: 21
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
WURCS=2.0/2,2,1/[a2122m-1b_1-5_2*NCC/3=O_4*NCC/3=O][a2112h-1a_1-5]/1-2/a3-b1WURCSPDB2Glycan 1.1.0
[]{[(3+1)][b-D-6-deoxy-GlcpNAc4NAc]{[(3+1)][a-D-Galp]{}}}LINUCSPDB-CARE
#3: Chemical...
ChemComp-OPE / PHOSPHORIC ACID MONO-(2-AMINO-ETHYL) ESTER / COLAMINE PHOSPHORIC ACID / Phosphorylethanolamine


Mass: 141.063 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: C2H8NO4P

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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: Neisseria gonorrhoeae Type IV pilin filament / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Neisseria gonorrhoeae (bacteria) / Strain: C30
Buffer solutionpH: 7.4 / Details: PBS 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 FIELDBright-field microscopy
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
9PHENIXmodel refinement
10Cootmodel refinement
11SPIDERinitial Euler assignment
12SPIDERfinal Euler assignment
13SPIDERclassification
14SPIDER3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 100.8 ° / Axial rise/subunit: 10.1 Å / Axial symmetry: C1
3D reconstructionResolution: 5.1 Å / Resolution method: OTHER / Num. of particles: 9855 / Details: model-map FSC 0.38 cut-off / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
RefinementHighest resolution: 5.1 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01726271
ELECTRON MICROSCOPYf_angle_d4.3735679
ELECTRON MICROSCOPYf_dihedral_angle_d8.56215456
ELECTRON MICROSCOPYf_chiral_restr0.5044179
ELECTRON MICROSCOPYf_plane_restr0.0074473

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