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- PDB-6o2t: Acetylated Microtubules -

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Basic information

Entry
Database: PDB / ID: 6o2t
TitleAcetylated Microtubules
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL 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 speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsEshun-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
OrganizationGrant numberCountry
National Science Foundation (United States)2016222703 United States
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 24, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
1A: Tubulin alpha-1B chain
1B: Tubulin alpha-1B chain
1C: Tubulin alpha-1B chain
1D: Tubulin alpha-1B chain
1E: Tubulin alpha-1B chain
1F: Tubulin alpha-1B chain
1G: Tubulin alpha-1B chain
1I: Tubulin alpha-1B chain
1J: Tubulin alpha-1B chain
1K: Tubulin alpha-1B chain
1L: Tubulin alpha-1B chain
1M: Tubulin alpha-1B chain
1N: Tubulin alpha-1B chain
1H: Tubulin beta chain
1O: Tubulin beta chain
1P: Tubulin beta chain
1Q: Tubulin beta chain
1R: Tubulin beta chain
1S: Tubulin beta chain
1T: Tubulin beta chain
1U: Tubulin beta chain
1V: Tubulin beta chain
1W: Tubulin beta chain
1X: Tubulin beta chain
1Y: Tubulin beta chain
1Z: Tubulin beta chain
2A: Tubulin alpha-1B chain
2B: Tubulin alpha-1B chain
2C: Tubulin alpha-1B chain
2D: Tubulin alpha-1B chain
2E: Tubulin alpha-1B chain
2F: Tubulin alpha-1B chain
2G: Tubulin alpha-1B chain
2I: Tubulin alpha-1B chain
2J: Tubulin alpha-1B chain
2K: Tubulin alpha-1B chain
2L: Tubulin alpha-1B chain
2M: Tubulin alpha-1B chain
2N: Tubulin alpha-1B chain
2H: Tubulin beta chain
2O: Tubulin beta chain
2P: Tubulin beta chain
2Q: Tubulin beta chain
2R: Tubulin beta chain
2S: Tubulin beta chain
2T: Tubulin beta chain
2U: Tubulin beta chain
2V: Tubulin beta chain
2W: Tubulin beta chain
2X: Tubulin beta chain
2Y: Tubulin beta chain
2Z: Tubulin beta chain
3A: Tubulin alpha-1B chain
3B: Tubulin alpha-1B chain
3C: Tubulin alpha-1B chain
3D: Tubulin alpha-1B chain
3E: Tubulin alpha-1B chain
3F: Tubulin alpha-1B chain
3G: Tubulin alpha-1B chain
3I: Tubulin alpha-1B chain
3J: Tubulin alpha-1B chain
3K: Tubulin alpha-1B chain
3L: Tubulin alpha-1B chain
3M: Tubulin alpha-1B chain
3N: Tubulin alpha-1B chain
3H: Tubulin beta chain
3O: Tubulin beta chain
3P: Tubulin beta chain
3Q: Tubulin beta chain
3R: Tubulin beta chain
3S: Tubulin beta chain
3T: Tubulin beta chain
3U: Tubulin beta chain
3V: Tubulin beta chain
3W: Tubulin beta chain
3X: Tubulin beta chain
3Y: Tubulin beta chain
3Z: Tubulin beta chain
4A: Tubulin alpha-1B chain
4B: Tubulin alpha-1B chain
4C: Tubulin alpha-1B chain
4D: Tubulin alpha-1B chain
4E: Tubulin alpha-1B chain
4F: Tubulin alpha-1B chain
4G: Tubulin alpha-1B chain
4I: Tubulin alpha-1B chain
4J: Tubulin alpha-1B chain
4K: Tubulin alpha-1B chain
4L: Tubulin alpha-1B chain
4M: Tubulin alpha-1B chain
4N: Tubulin alpha-1B chain
4H: Tubulin beta chain
4O: Tubulin beta chain
4P: Tubulin beta chain
4Q: Tubulin beta chain
4R: Tubulin beta chain
4S: Tubulin beta chain
4T: Tubulin beta chain
4U: Tubulin beta chain
4V: Tubulin beta chain
4W: Tubulin beta chain
4X: Tubulin beta chain
4Y: Tubulin beta chain
4Z: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)5,257,351260
Polymers5,205,835104
Non-polymers51,516156
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 52 / Rise per n subunits: 9.3 Å / Rotation per n subunits: -27.7 °)

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Components

#1: Protein/peptide ...
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 52 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Brain / References: UniProt: Q2XVP4
#2: Protein/peptide ...
Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 52 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Brain / References: UniProt: P02554
#3: Chemical...
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 52 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#4: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 52 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: Chemical...
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 52 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Acetylated Microtubule / Type: TISSUE / Entity ID: 1, 2 / Source: NATURAL
Source (natural)Organism: Sus scrofa (pig) / Tissue: Brain
Buffer solutionpH: 6.8
Details: Contains 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH (stored at 4 degrees Celsius).
Buffer component

Buffer-ID: 1

IDConc.NameFormula
180 mMPIPESC8H18N2O6S2
21 mMmagnesium chlorideMgCl2
31 mMEGTAC14H24N2O10
SpecimenConc.: 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 supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 310.15 K / Details: Blotted for 4 seconds at blot force 10.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Preliminary grid screening was performed manually and all of the alignments were initially done using a gold calibration grid.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage 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 opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 143 eV
Image scansWidth: 3840 / Height: 3710

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Processing

EM software
IDNameVersionCategory
1Appion2particle selection
2SerialEM3.7image acquisition
4CTFFIND4CTF correction
7PHENIX1.14model fitting
9PHENIX1.14model refinement
10EMAN1.9initial Euler assignment
11EMAN1.9final Euler assignment
12EMAN1.9classification
13FREALIGN9.113D reconstruction
CTF correctionDetails: CTFFIND4 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -27.7 ° / Axial rise/subunit: 9.3 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 20256 / Details: Extracted Helical Segments
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18432 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingB 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 buildingPDB-ID: 3JAR

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