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- PDB-2hil: Structure of the Neisseria gonorrhoeae Type IV pilus filament fro... -

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Basic information

Entry
Database: PDB / ID: 2hil
TitleStructure of the Neisseria gonorrhoeae Type IV pilus filament from x-ray crystallography and electron cryomicroscopy
ComponentsFimbrial protein
KeywordsCELL ADHESION / TYPE IV PILI / virulence factors / DNA binding protein / natural transformation / antigenic variation
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: 12.5 Å
AuthorsCraig, L. / Volkmann, N. / Egelman, E.H. / Tainer, J.A.
CitationJournal: Mol Cell / Year: 2006
Title: Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions.
Authors: Lisa Craig / Niels Volkmann / Andrew S Arvai / Michael E Pique / Mark Yeager / Edward H Egelman / John A Tainer /
Abstract: Type IV pili (T4P) are long, thin, flexible filaments on bacteria that undergo assembly-disassembly from inner membrane pilin subunits and exhibit astonishing multifunctionality. Neisseria ...Type IV pili (T4P) are long, thin, flexible filaments on bacteria that undergo assembly-disassembly from inner membrane pilin subunits and exhibit astonishing multifunctionality. Neisseria gonorrhoeae (gonococcal or GC) T4P are prototypic virulence factors and immune targets for increasingly antibiotic-resistant human pathogens, yet detailed structures are unavailable for any T4P. Here, we determined a detailed experimental GC-T4P structure by quantitative fitting of a 2.3 A full-length pilin crystal structure into a 12.5 A resolution native GC-T4P reconstruction solved by cryo-electron microscopy (cryo-EM) and iterative helical real space reconstruction. Spiraling three-helix bundles form the filament core, anchor the globular heads, and provide strength and flexibility. Protruding hypervariable loops and posttranslational modifications in the globular head shield conserved functional residues in pronounced grooves, creating a surprisingly corrugated pilus surface. These results clarify T4P multifunctionality and assembly-disassembly while suggesting unified assembly mechanisms for T4P, archaeal flagella, and type II secretion system filaments.
History
DepositionJun 29, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 12, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id
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.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _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.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _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.ptnr1_label_seq_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 / _struct_conn.ptnr2_label_seq_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)319,96754
Polymers309,78918
Non-polymers10,17836
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Fimbrial protein / Pilin / MS11 antigen


Mass: 17210.494 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Source: (natural) Neisseria gonorrhoeae (bacteria) / References: UniProt: P02974
#2: Polysaccharide
alpha-D-galactopyranose-(1-3)-2,4-bisacetamido-2,4-dideoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGalpa1-3DGlcpNAc[4NAc]b1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O_4*NCC/3=O][a2112h-1a_1-5]/1-2/a3-b1WURCSPDB2Glycan 1.1.0
[][D-1-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.: 18 / Source method: obtained synthetically / Formula: C2H8NO4P
Sequence detailsACCORDING TO AUTHORS THE RESIDUES HAVE BEEN CONFIRMED BY SEQUENCING THE PILE GENE THAT ENCODES THE ...ACCORDING TO AUTHORS THE RESIDUES HAVE BEEN CONFIRMED BY SEQUENCING THE PILE GENE THAT ENCODES THE GC PILIN SUBUNIT

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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: N. gonorrhoeae Type IV pili / Type: COMPLEX
Details: native pili were sheared from N. gonorrhoeae cells and purified by successive rounds of precipitation in neutral pH buffer, which causes the filaments to aggregate, followed by resuspension in high pH buffer
Buffer solutionName: 50 mM CHES / pH: 9.5 / Details: 50 mM CHES
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil holey carbon grids, glow discharged with amyl amine
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE
Details: 5 ul of sample were applied to grids for 1 minute, blotted for 2.5 seconds then plunge-frozen in liquid ethane using an FEI Vitrobot

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM200FEG / Date: Jun 23, 2004
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1100 nm
Specimen holderTemperature: 95 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 10 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 12
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1CoAnmodel fitting
2IHRSR3D reconstruction
CTF correctionDetails: Wiener filter
3D reconstructionMethod: IHRSR / Resolution: 12.5 Å / Num. of particles: 25000 / Nominal pixel size: 2.23676682 Å / Actual pixel size: 2.23676682 Å / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: real-space density correlation coefficient / Details: METHOD--CoAn REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 2HI2

2hi2

RefinementHighest resolution: 12.5 Å
Details: The GC pilus filament structure was built by computationally fitting a single GC pilin subunit (2HI2.pdb) into a cryoEM density map of the pilus filament (EMDB code xxx) using the program ...Details: The GC pilus filament structure was built by computationally fitting a single GC pilin subunit (2HI2.pdb) into a cryoEM density map of the pilus filament (EMDB code xxx) using the program CoAn (Volkmann and Hanein, J. Struct. Biol., 125, 176-184, 1999), and then applying the symmetry operators defined by the cryoEM reconstruction (10.5 Angstrom rise and a 100.8 degree azimuthal rotation, Craig et al., Mol. Cell xxx, yyy, 2006) to the subunit.
Refinement stepCycle: LAST / Highest resolution: 12.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21726 0 648 0 22374
NMR softwareName: CoAn / Classification: refinement
RefinementSoftware ordinal: 1
Details: The GC pilus filament structure was built by computationally fitting a single GC pilin subunit (2HI2.pdb) into a cryoEM density map of the pilus filament (EMDB code xxx) using the program ...Details: The GC pilus filament structure was built by computationally fitting a single GC pilin subunit (2HI2.pdb) into a cryoEM density map of the pilus filament (EMDB code xxx) using the program CoAn (Volkmann and Hanein, J. Struct. Biol., 125, 176-184, 1999), and then applying the symmetry operators defined by the cryoEM reconstruction (10.5 Angstrom rise and a 100.8 degree azimuthal rotation, Craig et al., Mol. Cell xxx, yyy, 2006) to the subunit.

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