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- EMDB-5762: Electron cryo-microscopy of four-stranded TubZ from B. thuringiensis -

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Basic information

Entry
Database: EMDB / ID: EMD-5762
TitleElectron cryo-microscopy of four-stranded TubZ from B. thuringiensis
Map dataReconstruction of four-stranded TubZ filament grown in the presence of GTP
Sample
  • Sample: Untagged full length TubZ from pBtoxis in Bacillus thuringiensis
  • Protein or peptide: ftsZ/tubulin-related protein
Keywordsplasmid segregation / tubulin family / bacterial cytoskeleton
Function / homology
Function and homology information


plasmid partitioning / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / GTPase activity / GTP binding / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin-like protein TubZ
Similarity search - Component
Biological speciesBacillus thuringiensis (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsMontabana EA / Agard DA
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis.
Authors: Elizabeth A Montabana / David A Agard /
Abstract: Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning ...Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments.
History
DepositionOct 2, 2013-
Header (metadata) releaseOct 23, 2013-
Map releaseFeb 19, 2014-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0155
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0155
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j4s
  • Surface level: 0.0155
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j4s
  • Surface level: 0.0155
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j4s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5762.jpg

Downloads & links

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Map

FileDownload / File: emd_5762.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of four-stranded TubZ filament grown in the presence of GTP
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.0155 / Movie #1: 0.0155
Minimum - Maximum-0.08206827 - 0.09640588
Average (Standard dev.)0.00018542 (±0.00950797)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.940.940.94
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z240.640240.640240.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.0820.0960.000

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Supplemental data

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Sample components

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Entire : Untagged full length TubZ from pBtoxis in Bacillus thuringiensis

EntireName: Untagged full length TubZ from pBtoxis in Bacillus thuringiensis
Components
  • Sample: Untagged full length TubZ from pBtoxis in Bacillus thuringiensis
  • Protein or peptide: ftsZ/tubulin-related protein

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Supramolecule #1000: Untagged full length TubZ from pBtoxis in Bacillus thuringiensis

SupramoleculeName: Untagged full length TubZ from pBtoxis in Bacillus thuringiensis
type: sample / ID: 1000
Oligomeric state: Helical filament with four protofilament strands
Number unique components: 1

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Macromolecule #1: ftsZ/tubulin-related protein

MacromoleculeName: ftsZ/tubulin-related protein / type: protein_or_peptide / ID: 1 / Name.synonym: TubZ-Bt, TubZ / Oligomeric state: helical four-stranded filament / Recombinant expression: Yes
Source (natural)Organism: Bacillus thuringiensis (bacteria) / Strain: serovar israelensis
Molecular weightExperimental: 54.3729 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Tubulin-like protein TubZ / InterPro: Tubulin/FtsZ, GTPase domain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.7
Details: 100 mM potassium acetate, 5 mM magnesium acetate, 50 mM HEPES
GridDetails: 400 mesh copper grid with holey carbon support, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Timed resolved state: GTP added to TubZ protein and incubated ~6 minutes before placing on grid.
Method: Blot 4.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at 250,000 times magnification
DateApr 22, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 414 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Whole micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 43.54512 Å
Applied symmetry - Helical parameters - Δ&Phi: 31.78843 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Spider
Details: Final Map has been low-pass filtered to 7 Angstrom and high-pass filtered to 30 Angstrom. A B-factor of -309 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~80 ...Details: Final Map has been low-pass filtered to 7 Angstrom and high-pass filtered to 30 Angstrom. A B-factor of -309 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~80 Angstrom has been applied.
DetailsThe particles were aligned using IHRSR.

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Atomic model buiding 1

Initial modelPDB ID:

2xkb


Chain - Chain ID: C
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j4s:
Helical Model of TubZ-Bt four-stranded filament

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