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- EMDB-8150: Structure and dynamics of single-isoform recombinant neuronal hum... -

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Basic information

Entry
Database: EMDB / ID: EMD-8150
TitleStructure and dynamics of single-isoform recombinant neuronal human tubulin
Map dataNone
Sample
  • Organelle or cellular component: Microtubules
    • Protein or peptide: Tubulin beta-3 chain
    • Protein or peptide: Tubulin alpha-1A chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
Keywordsmicrotubules / tubulin / single isoform / recombinant / dynamic instability / STRUCTURAL PROTEIN
Function / homology
Function and homology information


netrin receptor binding / Post-chaperonin tubulin folding pathway / axonemal microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / organelle transport along microtubule / glial cell differentiation / forebrain morphogenesis ...netrin receptor binding / Post-chaperonin tubulin folding pathway / axonemal microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / organelle transport along microtubule / glial cell differentiation / forebrain morphogenesis / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / neuron projection arborization / cytoskeleton-dependent intracellular transport / cerebellar cortex morphogenesis / Formation of tubulin folding intermediates by CCT/TriC / dentate gyrus development / COPI-independent Golgi-to-ER retrograde traffic / pyramidal neuron differentiation / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / centrosome cycle / motor behavior / COPI-dependent Golgi-to-ER retrograde traffic / response to L-glutamate / smoothened signaling pathway / intercellular bridge / regulation of synapse organization / startle response / locomotory exploration behavior / Recycling pathway of L1 / microtubule polymerization / microtubule-based process / RHO GTPases activate IQGAPs / response to tumor necrosis factor / Hedgehog 'off' state / COPI-mediated anterograde transport / response to mechanical stimulus / Activation of AMPK downstream of NMDARs / condensed chromosome / 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 / homeostasis of number of cells within a tissue / 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 / cellular response to calcium ion / adult locomotory behavior / filopodium / axon guidance / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / intracellular protein transport / neuron migration / peptide binding / synapse organization / neuromuscular junction / visual learning / PKR-mediated signaling / recycling endosome / cerebral cortex development / structural constituent of cytoskeleton / mitotic spindle / memory / 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 / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / lamellipodium / mitotic cell cycle / gene expression / growth cone / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron apoptotic process / microtubule / hydrolase activity / protein heterodimerization activity / cell division / axon / GTPase activity / neuronal cell body / dendrite / protein-containing complex binding / GTP binding / structural molecule activity / extracellular exosome / identical protein binding
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-3 chain / Tubulin alpha-1A chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsVemu A / Atherton J
Funding support United Kingdom, United States, 3 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: J Biol Chem / Year: 2016
Title: Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.
Authors: Annapurna Vemu / Joseph Atherton / Jeffrey O Spector / Agnieszka Szyk / Carolyn A Moores / Antonina Roll-Mecak /
Abstract: Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and ...Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.
History
DepositionApr 19, 2016-
Header (metadata) releaseMay 4, 2016-
Map releaseMay 4, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jco
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5jco
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8150.png

Downloads & links

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Map

FileDownload / File: emd_8150.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 208 pix.
= 253.76 Å		1.22 Å/pix.
x 80 pix.
= 97.6 Å		1.22 Å/pix.
x 166 pix.
= 202.52 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: 1.22 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.043 / Movie #1: 0.043
Minimum - Maximum-0.09062958 - 0.1718602
Average (Standard dev.)0.0035820229 (±0.015500975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions80166208
Spacing16680208
CellA: 202.52 Å / B: 97.600006 Å / C: 253.76001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z16680208
origin x/y/z0.0000.0000.000
length x/y/z202.52097.600253.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ329329329
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS16680208
D min/max/mean-0.0910.1720.004

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Supplemental data

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Sample components

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Entire : Microtubules

EntireName: Microtubules
Components
  • Organelle or cellular component: Microtubules
    • Protein or peptide: Tubulin beta-3 chain
    • Protein or peptide: Tubulin alpha-1A chain
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Microtubules

SupramoleculeName: Microtubules / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: 14pf GMPCPP-bound microtubule composed of human beta3 and alpha1a tubulin
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110 kDa/nm

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Macromolecule #1: Tubulin beta-3 chain

MacromoleculeName: Tubulin beta-3 chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.809926 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS IHQLVENTDE TYCIDNEALY DICFRTLKLA TPTYGDLNHL VSATMSGVTT SL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVATVF RGR MSMKEV DEQMLAIQSK NSSYFVEWIP NNVKVAVCDI PPRGLKMSST FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY Q

UniProtKB: Tubulin beta-3 chain

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Macromolecule #2: Tubulin alpha-1A chain

MacromoleculeName: Tubulin alpha-1A chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.649023 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
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 RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GV

UniProtKB: Tubulin alpha-1A 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
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.9 / Component - Name: BRB80
VitrificationCryogen name: ETHANE

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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

Final reconstructionApplied symmetry - Helical parameters - Δz: 8.86 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.71 °
Applied symmetry - Helical parameters - Axial symmetry: C14 (14 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER
Details: Overall map resolution was 4.2 Angstrom by gold-standard FSCtrue (Chen et al., 2013). The tubulin portion of the map was at higher resolution: at least 4 Angstrom according to the Blocres resolution measure.
Number images used: 10164
Startup modelType of model: OTHER / Details: Synthetic Kinesin Decorated Microtubule
Final angle assignmentType: NOT APPLICABLE

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