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- EMDB-1954: Negative stain reconstruction of the Vibrio cholerae toxin coregu... -

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Basic information

Entry
Database: EMDB / ID: EMD-1954
TitleNegative stain reconstruction of the Vibrio cholerae toxin coregulated pilus (TCP)
Map dataTCP - negative stain
Sample
  • Sample: Toxin coregulated pilus (TCP)
  • Protein or peptide: TcpA
KeywordsVibrio cholerae / Type IV pili / helical filaments / helical symmetry / autoagglutination
Biological speciesVibrio cholerae (bacteria)
Methodhelical reconstruction / negative staining / Resolution: 21.0 Å
AuthorsLi J / Egelman EH / Craig L
CitationJournal: J Mol Biol / Year: 2012
Title: Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus.
Authors: Juliana Li / Edward H Egelman / Lisa Craig /
Abstract: Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are ...Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8 M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-Å axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces.
History
DepositionAug 25, 2011-
Header (metadata) releaseSep 2, 2011-
Map releaseMar 22, 2012-
UpdateMar 22, 2012-
Current statusMar 22, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

em-1954.jpg

Downloads & links

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Map

FileDownload / File: emd_1954.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTCP - negative stain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 200 pix.
= 500. Å		2.5 Å/pix.
x 200 pix.
= 500. Å		2.5 Å/pix.
x 200 pix.
= 500. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.09328359 - 0.35152596
Average (Standard dev.)0.00434349 (±0.03336516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 500.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z500.000500.000500.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.0930.3520.004

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Supplemental data

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Sample components

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Entire : Toxin coregulated pilus (TCP)

EntireName: Toxin coregulated pilus (TCP)
Components
  • Sample: Toxin coregulated pilus (TCP)
  • Protein or peptide: TcpA

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Supramolecule #1000: Toxin coregulated pilus (TCP)

SupramoleculeName: Toxin coregulated pilus (TCP) / type: sample / ID: 1000 / Oligomeric state: Polymer / Number unique components: 1
Molecular weightExperimental: 42 MDa / Theoretical: 42 MDa

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Macromolecule #1: TcpA

MacromoleculeName: TcpA / type: protein_or_peptide / ID: 1 / Name.synonym: Type IV pilin / Oligomeric state: polymer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: RT4225 / Cell: Vibrio cholerae RT4225 / Location in cell: surface
Molecular weightExperimental: 21 KDa / Theoretical: 21 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.4
Details: PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 pH 7.4), 10 mM EDTA
StainingType: NEGATIVE / Details: 1% phosphotungstic acid, pH 7
GridDetails: carbon-coated copper grids (EMS)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: corrected at 100KX
DetailsLow dose mode
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON COOLSCAN / Digitization - Sampling interval: 2.49 µm / Number real images: 15 / Average electron dose: 10 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 30000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 30000
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 8.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 96.8 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IHRSR
Details: reconstruction was obtained from 8,034 overlapping particles (200 pixels length with a 190-pixel overlap, 80 pixels in width, padded to 200 pixels)

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Atomic model buiding 1

Initial modelPDB ID:

1oqv


Chain - Chain ID: A
DetailsPDBEntryID_givenInChain. Protocol: Rigid body. The TcpA pilin subunit was docked manually using the program Chimera. The filament was generated by applying the symmetry parameters to the docked subunit.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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