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Yorodumi- PDB-5n5n: Cryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb mi... -
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-Basic information
Entry | Database: PDB / ID: 5n5n | |||||||||
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Title | Cryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules | |||||||||
Components |
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Keywords | STRUCTURAL PROTEIN / Microtubules Dynamics Tubulin Isoform | |||||||||
Function / homology | Function and homology information odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC ...odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / natural killer cell mediated cytotoxicity / COPI-independent Golgi-to-ER retrograde traffic / Kinesins / Assembly and cell surface presentation of NMDA receptors / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / regulation of synapse organization / intercellular bridge / nuclear envelope lumen / Recycling pathway of L1 / RHOH GTPase cycle / spindle assembly / RHO GTPases activate IQGAPs / MHC class I protein binding / Hedgehog 'off' state / cytoplasmic microtubule / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / MHC class II antigen presentation / cellular response to interleukin-4 / AURKA Activation by TPX2 / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / azurophil granule lumen / Regulation of PLK1 Activity at G2/M Transition / double-stranded RNA binding / mitotic cell cycle / cell body / microtubule / Potential therapeutics for SARS / cytoskeleton / membrane raft / cell division / protein domain specific binding / GTPase activity / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / structural molecule activity / GTP binding / protein-containing complex / extracellular exosome / extracellular region / metal ion binding / nucleus / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
Authors | Vemu, A. / Atherton, J. / Spector, J.O. / Moores, C.A. / Roll-Mecak, A. | |||||||||
Funding support | United Kingdom, United States, 2items
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Citation | Journal: Mol Biol Cell / Year: 2017 Title: Tubulin isoform composition tunes microtubule dynamics. Authors: Annapurna Vemu / Joseph Atherton / Jeffrey O Spector / Carolyn A Moores / Antonina Roll-Mecak / Abstract: Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule- ...Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5n5n.cif.gz | 958.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5n5n.ent.gz | 813.8 KB | Display | PDB format |
PDBx/mmJSON format | 5n5n.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/n5/5n5n ftp://data.pdbj.org/pub/pdb/validation_reports/n5/5n5n | HTTPS FTP |
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-Related structure data
Related structure data | 3589MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS ensembles :
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-Components
#1: Protein | Mass: 47735.793 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Details: GMPCPP (GTP-analogue) bound / Source: (gene. exp.) Homo sapiens (human) / Cell line: tsA201 cells / Gene: TUBB, TUBB5, OK/SW-cl.56 / Cell line (production host): tsA201 cells / Production host: Homo sapiens (human) / References: UniProt: P07437 #2: Protein | Mass: 48665.027 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Details: GTP-bound / Source: (gene. exp.) Homo sapiens (human) / Cell line: tsA201 cells / Gene: TUBA1B / Cell line (production host): tsA201 cells / Production host: Homo sapiens (human) / References: UniProt: P68363 #3: Chemical | ChemComp-G2P / #4: Chemical | ChemComp-MG / #5: Chemical | ChemComp-GTP / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Human tsA201 cell tubulin microtubules / Type: COMPLEX Details: Microtubules formed from tsA201 cell tubulin (14pf) with bound GTP analogue GMPCPP. Tubulin was purified via TOG affinity. Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Homo sapiens (human) |
Buffer solution | pH: 6.8 Details: BRB80 with GMPCPP (80mM PIPES, 2mM MgCl2, 1mM EGTA, 1mM DTT, 1mM GMPCPP) |
Specimen | Conc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Microtubules polymerised at 25mg/ml in BRB80 with GMPCPP and diluted to a final concentration of 2.5mg/ml |
Specimen support | Grid material: COPPER / Grid type: C-flat-2/2 |
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) |
Image scans | Sampling size: 1.221 µm |
-Processing
Software | Name: REFMAC / Version: 5.8.0135 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13000 Details: Gold-standard high-resolution noise substitution test employed in resolution estimation (Chen et al., 2013) Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 4→214.9 Å / Cor.coef. Fo:Fc: 0.812 / SU B: 81.863 / SU ML: 0.956 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 66.218 Å2
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Refinement step | Cycle: 1 / Total: 40470 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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