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- EMDB-6390: Tubulin cofactors-D,E and Arl2-Q73L GTPase form a stable heterotr... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6390 | |||||||||
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Title | Tubulin cofactors-D,E and Arl2-Q73L GTPase form a stable heterotrimeric chaperone | |||||||||
![]() | Reconstruction of TBC-DEG(Q73L) | |||||||||
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![]() | Tubulin cofactors / Microtubule dynamics / Chaperones / tubulin biogenesis or degradation | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 24.0 Å | |||||||||
![]() | Nithianantham S / Le S / Seto E / Jia W / Leary J / Corbett KD / Moore JK / Al-Bassam J | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 7.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.3 KB 11.3 KB | Display Display | ![]() |
Images | ![]() ![]() | 42.5 KB 4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.8 KB | Display | ![]() |
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Full document | ![]() | 77.9 KB | Display | |
Data in XML | ![]() | 495 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of TBC-DEG(Q73L) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Tubulin cofactor complex (TBC-DEG)
Entire | Name: Tubulin cofactor complex (TBC-DEG) |
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Components |
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-Supramolecule #1000: Tubulin cofactor complex (TBC-DEG)
Supramolecule | Name: Tubulin cofactor complex (TBC-DEG) / type: sample / ID: 1000 Details: TBC-DEG complex behaves as a single biochemical entity. Oligomeric state: heterotrimeric / Number unique components: 3 |
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Molecular weight | Experimental: 205 KDa / Theoretical: 205 KDa / Method: SEC-MALS |
-Macromolecule #1: tubulin folding cofactor D
Macromolecule | Name: tubulin folding cofactor D / type: protein_or_peptide / ID: 1 / Name.synonym: TBCD / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: tubulin folding cofactor E
Macromolecule | Name: tubulin folding cofactor E / type: protein_or_peptide / ID: 2 / Name.synonym: TBCE / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #3: ARL2 GTPase, Q73L mutant
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7 Details: 50 mM HEPES, 100 mM KCl, 0.1 mM GTP, 3 mM b-mercaptoethanol |
Staining | Type: NEGATIVE Details: The proteins were incubated on carbon-coated grids, briefly washed, and stained with 1% uranyl formate. |
Grid | Details: glow-discharged 200 mesh gold grid with thin carbon support |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | JEOL 2100F |
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Date | Jun 10, 2014 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 3.5 µm / Number real images: 80 / Average electron dose: 9 e/Å2 / Bits/pixel: 5 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated 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 stage | Specimen holder model: JEOL |
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Image processing
Details | TBC-DEG particles were picked semi-automatically using e2boxer.py. |
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CTF correction | Details: Each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: Spider, EMAN2 / Number images used: 18000 |
Final two d classification | Number classes: 400 |