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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: EMD-3589
TitleCryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules
Map dataHEK cell tubulin microtubule cryo-EM reconstruction
Sample
  • Complex: Human tsA201 cell tubulin microtubules
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha-1B chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
KeywordsMicrotubules Dynamics Tubulin Isoform / structural protein
Function / homology
Function and homology information


odontoblast differentiation / Post-chaperonin tubulin folding pathway / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic ...odontoblast differentiation / Post-chaperonin tubulin folding pathway / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / Gap junction assembly / Kinesins / Assembly and cell surface presentation of NMDA receptors / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / natural killer cell mediated cytotoxicity / intercellular bridge / regulation of synapse organization / nuclear envelope lumen / cytoplasmic microtubule / MHC class I protein binding / Recycling pathway of L1 / microtubule-based process / RHOH GTPase cycle / spindle assembly / RHO GTPases activate IQGAPs / cellular response to interleukin-4 / Hedgehog 'off' state / 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 / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / AURKA Activation by TPX2 / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / cytoplasmic ribonucleoprotein granule / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / azurophil granule lumen / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / double-stranded RNA binding / mitotic cell cycle / cell body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / Potential therapeutics for SARS / cytoskeleton / membrane raft / protein domain specific binding / cell division / GTPase activity / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / GTP binding / structural molecule activity / protein-containing complex / extracellular exosome / extracellular region / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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
Similarity search - Domain/homology
Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsVemu A / Atherton J
Funding support United Kingdom, United States, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
Intramural Programs of the NINDS and the NHLBI, National Institutes of Health United States
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 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
DepositionFeb 14, 2017-
Header (metadata) releaseJun 21, 2017-
Map releaseNov 1, 2017-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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

emd_3589.png

Downloads & links

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Map

FileDownload / File: emd_3589.map.gz / Format: CCP4 / Size: 11.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHEK cell tubulin microtubule cryo-EM reconstruction
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0431 / Movie #1: 0.0431
Minimum - Maximum-0.11131498 - 0.19080862
Average (Standard dev.)0.00384792 (±0.016365934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions83172214
Spacing17283214
CellA: 210.012 Å / B: 101.342995 Å / C: 261.29398 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: Human tsA201 cell tubulin microtubules
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha-1B chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Human tsA201 cell tubulin microtubules

SupramoleculeName: Human tsA201 cell tubulin microtubules / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Microtubules formed from tsA201 cell tubulin (14pf) with bound GTP analogue GMPCPP. Tubulin was purified via TOG affinity.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 1 / Details: GMPCPP (GTP-analogue) bound / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.735793 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQVFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMAVT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY Q

UniProtKB: Tubulin beta chain

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Macromolecule #2: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 2 / Details: GTP-bound / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.665027 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GV

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #3: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 6.8
Details: BRB80 with GMPCPP (80mM PIPES, 2mM MgCl2, 1mM EGTA, 1mM DTT, 1mM GMPCPP)
GridModel: C-flat-2/2 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
DetailsMicrotubules polymerised at 25mg/ml in BRB80 with GMPCPP and diluted to a final concentration of 2.5mg/ml

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN
Details: Gold-standard high-resolution noise substitution test employed in resolution estimation (Chen et al., 2013)
Number images used: 13000
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-5n5n:
Cryo-EM structure of tsA201 cell alpha1B and betaI and betaIVb microtubules

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