|Entry||Database: PDB / ID: 6o2q|
|Keywords||STRUCTURAL PROTEIN / microtubule / cytoskeleton / acetylation|
|Function / homology|
Function and homology information
RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic ...RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / RHO GTPases Activate Formins / microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule / GTPase activity / GTP binding / cytoplasm
Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin ...Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site
Tubulin beta chain / Tubulin alpha-1B chain
|Biological species||Sus scrofa (pig)|
|Method||ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.7 Å|
|Authors||Eshun-Wilson, L. / Zhang, R. / Portran, D. / Nachury, M.V. / Toso, D. / Lohr, T. / Vendruscolo, M. / Bonomi, M. / Fraser, J.S. / Nogales, E.|
|Funding support|| United States, 1items |
|Citation||Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019|
Title: Effects of α-tubulin acetylation on microtubule structure and stability.
Authors: Lisa Eshun-Wilson / Rui Zhang / Didier Portran / Maxence V Nachury / Daniel B Toso / Thomas Löhr / Michele Vendruscolo / Massimiliano Bonomi / James S Fraser / Eva Nogales /
Abstract: Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule ...Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Tubulin alpha-1B chain
B: Tubulin beta chain
C: Tubulin alpha-1B chain
D: Tubulin beta chain
E: Tubulin alpha-1B chain
F: Tubulin beta chain
G: Tubulin beta chain
H: Tubulin beta chain
I: Tubulin beta chain
J: Tubulin alpha-1B chain
K: Tubulin alpha-1B chain
L: Tubulin alpha-1B chain
Mass: 50204.445 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Brain / References: UniProt: Q2XVP4
Mass: 49907.770 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Brain / References: UniProt: P02554
Mass: 523.180 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction|
|Component||Name: Acetylated Microtubule / Type: TISSUE / Entity ID: 1, 2 / Source: NATURAL|
|Source (natural)||Organism: Sus scrofa (pig) / Tissue: Brain|
|Buffer solution||pH: 6.8 |
Details: Contains 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH (stored at 4 degrees Celsius).
|Specimen||Conc.: 10 mg/ml|
Details: Acetylated and deacetylated tubulin preparations were produced by treating purified brain tubulin with the acetyltransferase TAT1 or the tubulin deacetylatase SIRT2 as done in Portran et al. Nat. Cell. Bio. 2017.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 310.15 K / Details: Blotted for 4 seconds at blot force 10.|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
Details: Preliminary grid screening was performed manually and all of the alignments were initially done using a gold calibration grid.
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 23364 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1422.3 nm / Calibrated defocus max: 2706.1 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K|
|Image recording||Average exposure time: 4 sec. / Electron dose: 25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 287|
|EM imaging optics||Energyfilter name: GIF Quantum LS / Energyfilter slit width: 143 eV|
|Image scans||Width: 3840 / Height: 3710|
|Software||Name: PHENIX / Version: 1.13_2998: / Classification: refinement|
|CTF correction||Details: CTFFIND4 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Helical symmerty||Angular rotation/subunit: -27.7 ° / Axial rise/subunit: 9.3 Å / Axial symmetry: C1|
|Particle selection||Num. of particles selected: 20256 / Details: Extracted Helical Segments|
|3D reconstruction||Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18432 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL|
|Atomic model building||B value: 126 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: 0.5 |
Details: We use PHENIX to perform real space refinement and sharpen our cryoEM maps.
|Atomic model building||PDB-ID: 3JAR|
|Refine LS restraints|
Refinement-ID: ELECTRON MICROSCOPY
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