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- EMDB-1760: Double filaments of C-terminally GFP-fused TubZ -

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Basic information

Entry
Database: EMDB / ID: EMD-1760
TitleDouble filaments of C-terminally GFP-fused TubZ
Map dataDouble helical filaments of a C-Terminal fusion of GFPmut2 to Bacillus thuringiensis serovar israelensis (ATCC 35646) TubZ (Q8KNP3_BACTI) Density for the GFP fusion of TubZ to remove positive density on the outer boundary of the negative stain and unconnected to the main helix.
Sample
  • Sample: Double filaments of TubZ-GFP
  • Protein or peptide: Cytomotive filament
KeywordsCytoskeleton / DNA segregation / FtsZ / FtsZ-like / pBtoxis / pbt156 / plasmid partitioning / RepX / tubulin / tubulin-like / TubZ
Biological speciesBacillus thuringiensis (bacteria)
Methodhelical reconstruction / negative staining / Resolution: 35.0 Å
AuthorsAylett CHS / Amos LA / Lowe J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2010
Title: Filament structure of bacterial tubulin homologue TubZ.
Authors: Christopher H S Aylett / Qing Wang / Katharine A Michie / Linda A Amos / Jan Löwe /
Abstract: Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a ...Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.
History
DepositionJul 9, 2010-
Header (metadata) releaseJul 23, 2010-
Map releaseOct 29, 2010-
UpdateApr 20, 2016-
Current statusApr 20, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 250
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 250
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1760.png

Downloads & links

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Map

FileDownload / File: emd_1760.map.gz / Format: CCP4 / Size: 666 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDouble helical filaments of a C-Terminal fusion of GFPmut2 to Bacillus thuringiensis serovar israelensis (ATCC 35646) TubZ (Q8KNP3_BACTI) Density for the GFP fusion of TubZ to remove positive density on the outer boundary of the negative stain and unconnected to the main helix.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
4 Å/pix.
x 86 pix.
= 344. Å		4.4 Å/pix.
x 45 pix.
= 198. Å		4.4 Å/pix.
x 45 pix.
= 198. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 4.4 Å / Y: 4.4 Å / Z: 4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 250.0 / Movie #1: 250
Minimum - Maximum-1466.819999999999936 - 1646.440000000000055
Average (Standard dev.)-760.423000000000002 (±830.966000000000008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions454586
Spacing454586
CellA: 198 Å / B: 198 Å / C: 344 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44
M x/y/z454586
origin x/y/z0.0000.0000.000
length x/y/z198.000198.000344.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS454586
D min/max/mean-1466.8201646.442-760.423

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Supplemental data

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Sample components

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Entire : Double filaments of TubZ-GFP

EntireName: Double filaments of TubZ-GFP
Components
  • Sample: Double filaments of TubZ-GFP
  • Protein or peptide: Cytomotive filament

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Supramolecule #1000: Double filaments of TubZ-GFP

SupramoleculeName: Double filaments of TubZ-GFP / type: sample / ID: 1000 / Details: Negatively stained / Oligomeric state: Dimer / Number unique components: 1

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Macromolecule #1: Cytomotive filament

MacromoleculeName: Cytomotive filament / type: protein_or_peptide / ID: 1 / Name.synonym: Cytomotive filament / Oligomeric state: Double filament / Recombinant expression: Yes
Source (natural)Organism: Bacillus thuringiensis (bacteria) / Strain: serovar israelensis / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5 / Details: 50 mM NaHEPES 7.5 150 mM KCl 5 mM MgCl2 1 mM GTPyS
StainingType: NEGATIVE / Details: 1% Uranyl Acetate
GridDetails: CuRh 300 mesh
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Bits/pixel: 8
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 67000
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: OTHER / Software - Name: MRC
Details: Final map was calculated from two averaged filaments

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