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- EMDB-3589: Cryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb mi... -

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Basic information

Entry
Database: EMDB / ID: 3589
TitleCryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules
Map dataHEK cell tubulin microtubule cryo-EM reconstruction
SampleHuman tsA201 cell tubulin microtubules:
Tubulin beta chain / Tubulin alpha-1B chain / (ligand) x 3
Function / homologyTubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal ...Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding 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. / Cilium Assembly / 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 / Formation of tubulin folding intermediates by CCT/TriC / Recycling pathway of L1 / Hedgehog 'off' state / Post-chaperonin tubulin folding pathway / Mitotic Prometaphase / Neutrophil degranulation / RHO GTPases Activate Formins / COPI-mediated anterograde transport / COPI-dependent Golgi-to-ER retrograde traffic / COPI-independent Golgi-to-ER retrograde traffic / Anchoring of the basal body to the plasma membrane / The role of GTSE1 in G2/M progression after G2 checkpoint / RHO GTPases activate IQGAPs / Intraflagellar transport / AURKA Activation by TPX2 / Carboxyterminal post-translational modifications of tubulin / Kinesins / 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 / cellular response to interleukin-4 / microtubule-based process / microtubule cytoskeleton organization / cytoplasmic ribonucleoprotein granule / ciliary basal body-plasma membrane docking / 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 / GTPase activity / cell division / membrane raft / myelin sheath / GTP binding / protein domain specific binding / ubiquitin protein ligase binding / structural molecule activity / neutrophil degranulation / extracellular exosome / extracellular region / nucleus / cytoplasm / Tubulin beta chain / Tubulin alpha-1B chain
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsVemu A / Atherton J
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 ReportPDB-ID: 5n5n

SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2017 / Header (metadata) release: Jun 21, 2017 / Map release: Nov 1, 2017 / Last update: Dec 13, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-5n5n
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3589.map.gz (map file in CCP4 format, 12221 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
214 pix
1.22 Å/pix.
= 261.294 Å
83 pix
1.22 Å/pix.
= 101.343 Å
172 pix
1.22 Å/pix.
= 210.012 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

(generated in cubic-lattice coordinate)

Voxel sizeX=Y=Z: 1.221 Å
Density
Contour Level:0.0431 (by author), 0.0431 (movie #1):
Minimum - Maximum-0.11131498 - 0.19080862
Average (Standard dev.)0.00384792 (0.016365934)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions83172214
Origin000
Limit82171213
Spacing17283214
CellA=B=C: 1 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2211.2211.221
M x/y/z17283214
origin x/y/z0.0000.0000.000
length x/y/z210.012101.343261.294
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS17283214
D min/max/mean-0.1110.1910.004

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Supplemental data

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Sample components

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Entire Human tsA201 cell tubulin microtubules

EntireName: Human tsA201 cell tubulin microtubules
Details: Microtubules formed from tsA201 cell tubulin (14pf) with bound GTP analogue GMPCPP. Tubulin was purified via TOG affinity.
Number of components: 6

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Component #1: protein, Human tsA201 cell tubulin microtubules

ProteinName: Human tsA201 cell tubulin microtubules
Details: Microtubules formed from tsA201 cell tubulin (14pf) with bound GTP analogue GMPCPP. Tubulin was purified via TOG affinity.
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #2: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Details: GMPCPP (GTP-analogue) bound / Recombinant expression: No
MassTheoretical: 47.735793 kDa
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: tsA201 cells

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Component #3: protein, Tubulin alpha-1B chain

ProteinName: Tubulin alpha-1B chain / Details: GTP-bound / Recombinant expression: No
MassTheoretical: 48.665027 kDa
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: tsA201 cells

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Component #4: ligand, PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

LigandName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.521208 kDa

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/ml
Buffer solution: BRB80 with GMPCPP (80mM PIPES, 2mM MgCl2, 1mM EGTA, 1mM DTT, 1mM GMPCPP)
pH: 6.8
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
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image acquisition

Image acquisitionSampling size: 1.221 microns

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 13000
3D reconstructionSoftware: FREALIGN / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Gold-standard high-resolution noise substitution test employed in resolution estimation (Chen et al., 2013)

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Atomic model buiding

Output model

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