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- EMDB-2447: Electron microscopy of the complex formed by chaperones TBCE and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2447
TitleElectron microscopy of the complex formed by chaperones TBCE and TBCB and alpha-tubulin
Map dataReconstruction of the complex formed by TBCE, TBCB and alpha-tubulin
Sample
  • Sample: Ternary complex of alpha-tubulin, TBCE and TBCB
  • Protein or peptide: Tubulin binding cofactor E
  • Protein or peptide: Tubulin binding cofactor B
  • Protein or peptide: alpha-tubulin
Keywordschaperones / protein folding / protein degradation / tubulin proteostasis
Function / homology
Function and homology information


peripheral nervous system neuron axonogenesis / post-chaperonin tubulin folding pathway / muscle atrophy / Post-chaperonin tubulin folding pathway / tubulin complex assembly / alpha-tubulin binding / developmental growth / cytoplasmic microtubule / cellular response to interleukin-4 / adult locomotory behavior ...peripheral nervous system neuron axonogenesis / post-chaperonin tubulin folding pathway / muscle atrophy / Post-chaperonin tubulin folding pathway / tubulin complex assembly / alpha-tubulin binding / developmental growth / cytoplasmic microtubule / cellular response to interleukin-4 / adult locomotory behavior / post-embryonic development / mitotic spindle organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / double-stranded RNA binding / protein folding / mitotic cell cycle / nervous system development / protein-folding chaperone binding / microtubule / cell differentiation / GTPase activity / ubiquitin protein ligase binding / GTP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Tubulin-specific chaperone E / TBCE, ubiquitin-like (Ubl) domain / Ubiquitin-like domain / CAP-Gly domain signature. / CAP Gly-rich domain / CAP Gly-rich domain / CAP Gly-rich domain superfamily / CAP-Gly domain / CAP-Gly domain profile. / CAP_GLY ...Tubulin-specific chaperone E / TBCE, ubiquitin-like (Ubl) domain / Ubiquitin-like domain / CAP-Gly domain signature. / CAP Gly-rich domain / CAP Gly-rich domain / CAP Gly-rich domain superfamily / CAP-Gly domain / CAP-Gly domain profile. / CAP_GLY / Alpha tubulin / Alpha tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / Leucine-rich repeat domain superfamily / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Tubulin alpha-1B chain / Tubulin-specific chaperone E / Tubulin-folding cofactor B
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsSerna M / Carranza G / Martin-Benito J / Janowsk R / Canals A / Coll M / Zabala JC / Valpuesta JM
CitationJournal: J Cell Sci / Year: 2015
Title: The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.
Authors: Marina Serna / Gerardo Carranza / Jaime Martín-Benito / Robert Janowski / Albert Canals / Miquel Coll / Juan Carlos Zabala / José María Valpuesta /
Abstract: Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation ...Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
History
DepositionAug 27, 2013-
Header (metadata) releaseSep 18, 2013-
Map releaseSep 10, 2014-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.051
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.051
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2447.png

Downloads & links

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Map

FileDownload / File: emd_2447.map.gz / Format: CCP4 / Size: 126 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the complex formed by TBCE, TBCB and alpha-tubulin
Voxel sizeX=Y=Z: 4.66 Å
Density
Contour LevelBy AUTHOR: 0.051 / Movie #1: 0.051
Minimum - Maximum-0.03980095 - 0.19663683
Average (Standard dev.)0.00350237 (±0.02621287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions323232
Spacing323232
CellA=B=C: 149.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.664.664.66
M x/y/z323232
origin x/y/z0.0000.0000.000
length x/y/z149.120149.120149.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS323232
D min/max/mean-0.0400.1970.004

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Supplemental data

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Sample components

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Entire : Ternary complex of alpha-tubulin, TBCE and TBCB

EntireName: Ternary complex of alpha-tubulin, TBCE and TBCB
Components
  • Sample: Ternary complex of alpha-tubulin, TBCE and TBCB
  • Protein or peptide: Tubulin binding cofactor E
  • Protein or peptide: Tubulin binding cofactor B
  • Protein or peptide: alpha-tubulin

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Supramolecule #1000: Ternary complex of alpha-tubulin, TBCE and TBCB

SupramoleculeName: Ternary complex of alpha-tubulin, TBCE and TBCB / type: sample / ID: 1000 / Oligomeric state: heterotrimer / Number unique components: 3
Molecular weightTheoretical: 137 KDa

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Macromolecule #1: Tubulin binding cofactor E

MacromoleculeName: Tubulin binding cofactor E / type: protein_or_peptide / ID: 1 / Name.synonym: Tubulin-specific chaperone E / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightTheoretical: 59 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac-1
SequenceUniProtKB: Tubulin-specific chaperone E / InterPro: CAP Gly-rich domain

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Macromolecule #2: Tubulin binding cofactor B

MacromoleculeName: Tubulin binding cofactor B / type: protein_or_peptide / ID: 2
Name.synonym: Tubulin-specific chaperone B, Tubulin-folding cofactor B
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) pLysS / Recombinant plasmid: pET3a
SequenceUniProtKB: Tubulin-folding cofactor B / InterPro: CAP Gly-rich domain

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Macromolecule #3: alpha-tubulin

MacromoleculeName: alpha-tubulin / type: protein_or_peptide / ID: 3 / Name.synonym: Tubulin alpha-1B chain / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Bovin / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceUniProtKB: Tubulin alpha-1B chain / InterPro: Alpha tubulin

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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

Concentration0.02 mg/mL
BufferpH: 6.7 / Details: 100mM MES-NaOH, 25mM KCl, 1mM MgCl2, 1mM EGTA
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 1 min.
GridDetails: 300 mesh copper grid with thin carbon support, glow discharged in air atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1200EXII
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 5.6 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateNov 1, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 85 / Average electron dose: 10 e/Å2

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Image processing

CTF correctionDetails: N. Grigorieff CTFFIND3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: EMAN, Spider, XMIPP
Details: Individual particles were manually selected using XMIPP software package.
Number images used: 26129
DetailsThe particles were manually selected using the XMIPP program

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Atomic model buiding 1

Initial modelPDB ID:

1v6e


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domain was fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1whg


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domain was fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

1tub


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domain was fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:

4icu


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domain was fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 5

Initial modelPDB ID:

3rj0


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domain was fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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