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- EMDB-1236: Type IV pilus structure by cryo-electron microscopy and crystallo... -

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Basic information

Entry
Database: EMDB / ID: EMD-1236
TitleType IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions.
Map dataCryoEM map for Neisseria gonorrhoeae Type IV pilus
Sample
  • Sample: Type IV pilus filament from Neisseria gonorrhoeae
  • Organelle or cellular component: N. gonorrhoeae Type IV pilus
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
Type IV major pilin protein PilE1
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 12.5 Å
AuthorsCraig L / Egelman EH / Volkmann N / Tainer JA
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, 2006-
Header (metadata) releaseJul 7, 2006-
Map releaseSep 1, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2hil
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2hil
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1236.gif

Downloads & links

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Map

FileDownload / File: emd_1236.map.gz / Format: CCP4 / Size: 283.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map for Neisseria gonorrhoeae Type IV pilus
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour Level1: 2.05 / Movie #1: 0.8
Minimum - Maximum-0.810034 - 3.94621
Average (Standard dev.)-0.0000000204282 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-18-18-26
Dimensions373754
Spacing373754
CellA: 101.6 Å / B: 101.6 Å / C: 152.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z404060
origin x/y/z0.0000.0000.000
length x/y/z101.600101.600152.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-18-18-26
NC/NR/NS373754
D min/max/mean-0.8103.946-0.000

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Supplemental data

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Sample components

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Entire : Type IV pilus filament from Neisseria gonorrhoeae

EntireName: Type IV pilus filament from Neisseria gonorrhoeae
Components
  • Sample: Type IV pilus filament from Neisseria gonorrhoeae
  • Organelle or cellular component: N. gonorrhoeae Type IV pilus

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Supramolecule #1000: Type IV pilus filament from Neisseria gonorrhoeae

SupramoleculeName: Type IV pilus filament from Neisseria gonorrhoeae / type: sample / ID: 1000 / Details: filaments are variable lengths - 1 to 4 microns
Oligomeric state: thousands of pilin subunits form a helical filament
Number unique components: 1

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Supramolecule #1: N. gonorrhoeae Type IV pilus

SupramoleculeName: N. gonorrhoeae Type IV pilus / type: organelle_or_cellular_component / ID: 1 / Name.synonym: pilus, GC-T4P
Details: GC-T4P are variable in length and contail thousands of copies of the pilin subunit. A 1-micron length would contain approx. 952 subunits, with a molecular weight of 17.7 kDa/subunit, giving ...Details: GC-T4P are variable in length and contail thousands of copies of the pilin subunit. A 1-micron length would contain approx. 952 subunits, with a molecular weight of 17.7 kDa/subunit, giving a total molecular weight of approx. 16.8 mDa.
Number of copies: 1 / Oligomeric state: helical filament / Recombinant expression: Yes
Source (natural)Organism: Neisseria gonorrhoeae (bacteria) / Strain: C30 / synonym: bacteria / Cell: N. gonorrhoeae / Organelle: pilus / Location in cell: outer membrane
Recombinant expressionOrganism: N. gonorrhoeae

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 9.5 / Details: 50 mM CHES, pH 9.5
GridDetails: Quantifoil holey carbon grids, glow-discharged w/ amyl amine
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4.2 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 2.5 sec. before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 4.5 K / Max: 4.8 K / Average: 4.5 K
Alignment procedureLegacy - Astigmatism: corrected at 135,000X magnification
DetailsImages were collected in low dose mode.
DateJun 23, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 12.7 µm / Number real images: 12 / Average electron dose: 10 e/Å2 / Bits/pixel: 14

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 10.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 100.8 °
Resolution.type: BY AUTHOR / Resolution: 12.5 Å / Resolution method: OTHER / Software - Name: IHRSR
Details: The final map was calculated from ~25,000 overlapping particles (508 Angstrom segments with 95% overlap)
DetailsNative Type IV pili were isoated by shearing from N. gonorrhoeae cells.

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Atomic model buiding 1

Initial modelPDB ID:

2hi2

SoftwareName: CoAn
DetailsProtocol: rigid body. A single asymmetric unit was isolated from the filament reconstruction using the watershed transform (Volkmann, J Struct Biol 138, 123-129, 2002) and used for unconstrained docking of a single pilin molecule. After fitting the subunit and builiding the filament model (PDB code 2HIL) the density map was corrected by scaling the amplitudes to the spherically averaged molecular transform of the filament model to minimize artifacts from amplitude distortion caused by the CTF and other experimental factors. This correction only affects visual appearance and not the subsequent automated docking process, which automatically corrects for amplitude distortions.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Target criteria: real-space density correlation coefficient between the experimental density and the density calculated from the search model
Output model

PDB-2hil:
Structure of the Neisseria gonorrhoeae Type IV pilus filament from x-ray crystallography and electron cryomicroscopy

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