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- PDB-6o2r: Deacetylated Microtubules -

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Basic information

Entry
Database: PDB / ID: 6o2r
TitleDeacetylated Microtubules
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / microtubule / cytoskeleton / acetylation
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / COPI-mediated anterograde transport / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 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 (NSF, 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.
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
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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Assembly

Deposited unit
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)606,61730
Polymers600,67312
Non-polymers5,94418
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52900 Å2
ΔGint-277 kcal/mol
Surface area161440 Å2

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Components

#1: Protein
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous 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
#2: Protein
Tubulin beta chain / Beta-tubulin


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
#3: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / 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: Deacetylated 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
IDConc.NameFormulaBuffer-ID
180 mMPIPESC8H18N2O6S21
21 mMmagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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. ...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.
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 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder 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

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
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: 29396 / Details: Extracted Helical Segments
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24692 / 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

3jar


Accession code: 3JAR / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00641796
ELECTRON MICROSCOPYf_angle_d0.75656784
ELECTRON MICROSCOPYf_dihedral_angle_d7.75224852
ELECTRON MICROSCOPYf_chiral_restr0.0526216
ELECTRON MICROSCOPYf_plane_restr0.0077398

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