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- EMDB-1495: Structural Analysis of the Saf Pilus by Electron Microscopy and I... -

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Basic information

Entry
Database: EMDB / ID: EMD-1495
TitleStructural Analysis of the Saf Pilus by Electron Microscopy and Image Processing. 3D reconstruction of the Type B Saf pilus, one of two conformations of the pilus refined.
Map data3D reconstruction of the Type B Saf pilus
Sample
  • Sample: Saf pilus
  • Protein or peptide: Wild-type SafA
KeywordsPilus / electron microscopy / image processing / single-particle analysis / atomic structure fitting
Function / homologySaf-pilin pilus formation protein / Saf-pilin pilus formation protein / Dr-adhesin superfamily / Adhesion domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Outer membrane protein
Function and homology information
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsSalih O / Remaut H / Waksman G / Orlova EV
CitationJournal: J Mol Biol / Year: 2008
Title: Structural analysis of the Saf pilus by electron microscopy and image processing.
Authors: Osman Salih / Han Remaut / Gabriel Waksman / Elena V Orlova /
Abstract: Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella ...Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a "beads on a string"-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.
History
DepositionMar 11, 2008-
Header (metadata) releaseMar 11, 2008-
Map releaseMar 31, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3crf
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1495.gif

Downloads & links

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Map

FileDownload / File: emd_1495.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the Type B Saf pilus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.59 Å/pix.
x 128 pix.
= 203.648 Å		1.59 Å/pix.
x 128 pix.
= 203.648 Å		1.59 Å/pix.
x 128 pix.
= 203.648 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.591 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.59 / Movie #1: 2
Minimum - Maximum-3.33278 - 86.760300000000001
Average (Standard dev.)0.0449852 (±2.95725)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 203.648 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5911.5911.591
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z203.648203.648203.648
α/β/γ90.00090.00090.000
start NX/NY/NZ-55-55-55
NX/NY/NZ111111111
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-3.33386.7600.045

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Supplemental data

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Sample components

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Entire : Saf pilus

EntireName: Saf pilus
Components
  • Sample: Saf pilus
  • Protein or peptide: Wild-type SafA

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Supramolecule #1000: Saf pilus

SupramoleculeName: Saf pilus / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 14 KDa

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Macromolecule #1: Wild-type SafA

MacromoleculeName: Wild-type SafA / type: protein_or_peptide / ID: 1 / Name.synonym: SafA pilus subunit / Oligomeric state: Fibre / Recombinant expression: Yes
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Strain: LT2 / Location in cell: Cell surface
Molecular weightExperimental: 14.4 KDa
Recombinant expressionOrganism: Escherichia coli C600 / Recombinant plasmid: pTrc 99a

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8 / Details: 10 mM TRIS-HCl (pH 8) and 10 mM NaCl
StainingType: NEGATIVE / Details: 2% ammonium molybdate for 5 seconds
GridDetails: Continuous-carbon film
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 1.591 µm / Number real images: 140 / Average electron dose: 25 e/Å2 / Od range: 1.5 / Bits/pixel: 8
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.4 mm / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 44000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic / Number images used: 8500
Final angle assignmentDetails: Imagic: alpha, beta, gamma
Final two d classificationNumber classes: 424

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Atomic model buiding 1

Initial modelPDB ID:

2co4

SoftwareName: URO
DetailsProtocol: Rigid body
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-3crf:
Electron Microscopy model of the Saf Pilus- Type B

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