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- EMDB-6392: Structural basis for TBC-DEG assembly with soluble tubulin dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-6392
TitleStructural basis for TBC-DEG assembly with soluble tubulin dimer
Map dataReconstruction of TBC-DEG with tubulin dimer
Sample
  • Sample: Tubulin cofactor complex [TBC-DE(N-GFP)G]
  • Protein or peptide: tubulin folding cofactor D
  • Protein or peptide: tubulin folding cofactor E
  • Protein or peptide: ARL2 GTPase, Q73L mutant
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
KeywordsTubulin cofactors / Microtubule dynamics / Chaperones / tubulin biogenesis or degradation / tubulin dimer / TBC-DEG
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsNithianantham S / Le S / Seto E / Jia W / Leary J / Corbett KD / Moore JK / Al-Bassam J
CitationJournal: Elife / Year: 2015
Title: Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.
Authors: Stanley Nithianantham / Sinh Le / Elbert Seto / Weitao Jia / Julie Leary / Kevin D Corbett / Jeffrey K Moore / Jawdat Al-Bassam /
Abstract: Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into ...Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.
History
DepositionJul 18, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseSep 23, 2015-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0163
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0163
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6392.gif

Downloads & links

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Map

FileDownload / File: emd_6392.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TBC-DEG with tubulin dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 128 pix.
= 256. Å		2 Å/pix.
x 128 pix.
= 256. Å		2 Å/pix.
x 128 pix.
= 256. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0163 / Movie #1: 0.0163
Minimum - Maximum-0.00000067 - 0.10691054
Average (Standard dev.)0.00117101 (±0.00657905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0000.1070.001

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Supplemental data

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Sample components

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Entire : Tubulin cofactor complex [TBC-DE(N-GFP)G]

EntireName: Tubulin cofactor complex [TBC-DE(N-GFP)G]
Components
  • Sample: Tubulin cofactor complex [TBC-DE(N-GFP)G]
  • Protein or peptide: tubulin folding cofactor D
  • Protein or peptide: tubulin folding cofactor E
  • Protein or peptide: ARL2 GTPase, Q73L mutant
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin

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Supramolecule #1000: Tubulin cofactor complex [TBC-DE(N-GFP)G]

SupramoleculeName: Tubulin cofactor complex [TBC-DE(N-GFP)G] / type: sample / ID: 1000
Details: TBC-DEG complex with tubulin dimer behaves as a single biochemical entity.
Oligomeric state: One heterotrimer of TBC-DEG binds to tubulin dimer
Number unique components: 5
Molecular weightExperimental: 308 KDa / Theoretical: 310 KDa / Method: SEC-MALS

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Macromolecule #1: tubulin folding cofactor D

MacromoleculeName: tubulin folding cofactor D / type: protein_or_peptide / ID: 1 / Name.synonym: TBCD / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDEST

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Macromolecule #2: tubulin folding cofactor E

MacromoleculeName: tubulin folding cofactor E / type: protein_or_peptide / ID: 2 / Name.synonym: TBCE / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDEST

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Macromolecule #3: ARL2 GTPase, Q73L mutant

MacromoleculeName: ARL2 GTPase, Q73L mutant / type: protein_or_peptide / ID: 3 / Name.synonym: DEG(Q73L) / Details: Arl2 locked mutant (Q73L) in a GTP-like state / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDEST

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Macromolecule #4: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 4 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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Macromolecule #5: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 5 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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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

Concentration0.5 mg/mL
BufferpH: 7
Details: 50 mM HEPES, 100 mM KCl, 0.1 mM GTP, 3 mM b-mercaptoethanol
StainingType: NEGATIVE
Details: The proteins were incubated on carbon-coated grids, briefly washed, and stained with 1% uranyl formate.
GridDetails: glow-discharged 200 mesh gold grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
DateJul 10, 2014
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 3.5 µm / Number real images: 90 / Average electron dose: 9 e/Å2 / Bits/pixel: 5
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50050 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsTBC-DEG:tubulin complex particles were picked semi-automatically using e2boxer.py.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: Spider, EMAN2 / Number images used: 20000
Final two d classificationNumber classes: 400

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