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- 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
TitleCryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / Microtubules Dynamics Tubulin Isoform / structural protein
Function / homologyBeta tubulin, autoregulation binding site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal ...Beta tubulin, autoregulation binding site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ family, GTPase domain / Tubulin C-terminal domain / Tubulin-beta mRNA autoregulation signal. / Post-chaperonin tubulin folding pathway / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Gap junction assembly / MHC class II antigen presentation / Separation of Sister Chromatids / Resolution of Sister Chromatid Cohesion / Regulation of PLK1 Activity at G2/M Transition / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Recycling pathway of L1 / Hedgehog 'off' state / Cilium Assembly / RHO GTPases Activate Formins / Kinesins / Carboxyterminal post-translational modifications of tubulin / AURKA Activation by TPX2 / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / Neutrophil degranulation / Formation of tubulin folding intermediates by CCT/TriC / Anchoring of the basal body to the plasma membrane / Intraflagellar transport / RHO GTPases activate IQGAPs / natural killer cell mediated cytotoxicity / cytoskeleton-dependent intracellular transport / cellular process / spindle assembly / GTPase activating protein binding / MHC class I protein binding / nuclear envelope lumen / cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / microtubule cytoskeleton organization / ciliary basal body-plasma membrane docking / cytoplasmic ribonucleoprotein granule / structural constituent of cytoskeleton / microtubule cytoskeleton / cell body / double-stranded RNA binding / azurophil granule lumen / regulation of G2/M transition of mitotic cell cycle / G2/M transition of mitotic cell cycle / microtubule / cytoskeleton / protein-containing complex binding / GTPase activity / membrane raft / myelin sheath / cell division / GTP binding / ubiquitin protein ligase binding / protein domain specific binding / structural molecule activity / neutrophil degranulation / extracellular exosome / extracellular region / nucleus / cytoplasm / Tubulin beta chain / Tubulin alpha-1B chain
Function and homology information
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsVemu, A. / Atherton, J. / Spector, J.O. / Moores, C.A. / Roll-Mecak, A.
CitationJournal: 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 ...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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2017 / Release: Nov 1, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 1, 2017Structure modelrepositoryInitial release
1.1Dec 13, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-3589
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Tubulin beta chain
K: Tubulin alpha-1B chain
G: Tubulin alpha-1B chain
H: Tubulin alpha-1B chain
I: Tubulin alpha-1B chain
J: Tubulin alpha-1B chain
L: Tubulin alpha-1B chain
A: Tubulin beta chain
C: Tubulin beta chain
D: Tubulin beta chain
E: Tubulin beta chain
F: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)584,96336
Polyers578,40512
Non-polymers6,55824
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)54520
ΔGint (kcal/M)-306
Surface area (Å2)176740
MethodPISA

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Components

#1: Protein/peptide
Tubulin beta chain / Tubulin beta-5 chain


Mass: 47735.793 Da / Num. of mol.: 6 / 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/peptide
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 48665.027 Da / Num. of mol.: 6 / 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 / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 6 / Formula: C11H18N5O13P3 / Comment: GMP-CPP (energy-carrying molecule analogue) *YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Formula: Mg / Magnesium
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 6 / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionDetails: BRB80 with GMPCPP (80mM PIPES, 2mM MgCl2, 1mM EGTA, 1mM DTT, 1mM GMPCPP)
pH: 6.8
SpecimenConc.: 2.5 mg/ml
Details: Microtubules polymerised at 25mg/ml in BRB80 with GMPCPP and diluted to a final concentration of 2.5mg/ml
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k)
Image scansSampling size: 1.221 microns

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Processing

SoftwareName: REFMAC / Version: 5.8.0135 / Classification: refinement
EM software
IDNameCategory
1EMANparticle selection
2SerialEMimage acquisition
7UCSF Chimeramodel fitting
10FREALIGNfinal Euler assignment
12FREALIGN3D reconstruction
13Cootmodel refinement
14PHENIXmodel refinement
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 13000
Details: Gold-standard high-resolution noise substitution test employed in resolution estimation (Chen et al., 2013)
Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcDetailsR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLSolvent ion probe radiiSolvent shrinkage radiiSolvent vdw probe radiiStereochemistry target valuesSolvent model details
166.218-5.481.19-0.851.441.474.040.812HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.440920.440924.00214.90102758100.0081.8630.9560.800.801.20MAXIMUM LIKELIHOOD WITH PHASESMASK
ELECTRON MICROSCOPY
Number of atoms included #1Total: 40470
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01941382
ELECTRON MICROSCOPYr_bond_other_d0.0010.02038220
ELECTRON MICROSCOPYr_angle_refined_deg1.1351.95956220
ELECTRON MICROSCOPYr_angle_other_deg0.8673.00087870
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.9425.0005094
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.96624.1231950
ELECTRON MICROSCOPYr_dihedral_angle_3_deg11.09715.0006684
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.64215.000252
ELECTRON MICROSCOPYr_chiral_restr0.0650.2006174
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.02147094
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0209738
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it0.7116.70220466
ELECTRON MICROSCOPYr_mcbond_other0.7116.70220465
ELECTRON MICROSCOPYr_mcangle_it1.34910.05225530
ELECTRON MICROSCOPYr_mcangle_other1.34910.05225531
ELECTRON MICROSCOPYr_scbond_it0.3276.73120916
ELECTRON MICROSCOPYr_scbond_other0.3276.73120909
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other0.71710.05630681
ELECTRON MICROSCOPYr_long_range_B_refined2.97956.28657454
ELECTRON MICROSCOPYr_long_range_B_other2.97856.28757451
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints ncs

Refine ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0 Å / Weight position: 0.05

Dom IDAuth asym IDEns IDNumber
1B149120
2A149120
1B249118
2C249118
1B349116
2D349116
1B449120
2E449120
1B549120
2F549120
1K649634
2G649634
1K749634
2H749634
1K849634
2I849634
1K949634
2J949634
1K1049634
2L1049634
1G1149636
2H1149636
1G1249634
2I1249634
1G1349634
2J1349634
1G1449634
2L1449634
1H1549634
2I1549634
1H1649634
2J1649634
1H1749634
2L1749634
1I1849634
2J1849634
1I1949634
2L1949634
1J2049634
2L2049634
1A2149122
2C2149122
1A2249118
2D2249118
1A2349120
2E2349120
1A2449124
2F2449124
1C2549118
2D2549118
1C2649118
2E2649118
1C2749122
2F2749122
1D2849116
2E2849116
1D2949118
2F2949118
1E3049120
2F3049120
Refine LS shellHighest resolution: 4 Å / R factor R free: 0 / R factor R work: 0.548 / Lowest resolution: 4.104 Å / Number reflection R free: 0 / Number reflection R work: 7593 / Total number of bins used: 20 / Percent reflection obs: 1

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