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- EMDB-3254: Subtomogram average of a piliated type IVa pilus machine in Myxoc... -

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Basic information

Entry
Database: EMDB / ID: EMD-3254
TitleSubtomogram average of a piliated type IVa pilus machine in Myxococcus xanthus cells with PilO tagged with a superfolder GFP
Map dataSubtomogram average of a piliated type IVa pilus machine in Myxococcus xanthus cells with PilO tagged with a superfolder GFP
Sample
  • Sample: Myxococcus xanthus DK1622 cells with PilO tagged with a superfolder GFP
  • Organelle or cellular component: Type IVa pilus
Keywordstype IV pilus / motor / motility / adhesion
Biological speciesMyxococcus xanthus (bacteria)
Methodsubtomogram averaging / cryo EM
AuthorsChang YW / Rettberg LA / Treuner-Lange A / Iwasa J / Sogaard-Andersen L / Jensen GJ
CitationJournal: Science / Year: 2016
Title: Architecture of the type IVa pilus machine.
Authors: Yi-Wei Chang / Lee A Rettberg / Anke Treuner-Lange / Janet Iwasa / Lotte Søgaard-Andersen / Grant J Jensen /
Abstract: Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of ...Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.
History
DepositionNov 22, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseMar 23, 2016-
UpdateMar 23, 2016-
Current statusMar 23, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.093
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.093
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3254.tif

emd_3254_1.tif

Downloads & links

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Map

FileDownload / File: emd_3254.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of a piliated type IVa pilus machine in Myxococcus xanthus cells with PilO tagged with a superfolder GFP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.8 Å/pix.
x 80 pix.
= 624. Å		7.8 Å/pix.
x 140 pix.
= 1092. Å		7.8 Å/pix.
x 80 pix.
= 624. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 7.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.093 / Movie #1: 0.093
Minimum - Maximum-0.20373002 - 0.26392484
Average (Standard dev.)0.0000034 (±0.09009819)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-60-60
Dimensions1408080
Spacing1408080
CellA: 624.0 Å / B: 1092.0 Å / C: 624.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.87.87.8
M x/y/z8014080
origin x/y/z0.0000.0000.000
length x/y/z624.0001092.000624.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-60-30-60
NC/NR/NS8014080
D min/max/mean-0.2040.2640.000

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Supplemental data

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Sample components

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Entire : Myxococcus xanthus DK1622 cells with PilO tagged with a superfold...

EntireName: Myxococcus xanthus DK1622 cells with PilO tagged with a superfolder GFP
Components
  • Sample: Myxococcus xanthus DK1622 cells with PilO tagged with a superfolder GFP
  • Organelle or cellular component: Type IVa pilus

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Supramolecule #1000: Myxococcus xanthus DK1622 cells with PilO tagged with a superfold...

SupramoleculeName: Myxococcus xanthus DK1622 cells with PilO tagged with a superfolder GFP
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Type IVa pilus

SupramoleculeName: Type IVa pilus / type: organelle_or_cellular_component / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Myxococcus xanthus (bacteria) / Strain: DK1622

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GATAN / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateMar 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 150 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 27500
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Software - Name: IMOD, TOMO3D, PEET / Number subtomograms used: 50

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