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Basic information

Entry
Database: PDB / ID: 5jco
TitleStructure and dynamics of single-isoform recombinant neuronal human tubulin
Components
  • Tubulin alpha-1A chain
  • Tubulin beta-3 chain
KeywordsSTRUCTURAL PROTEIN / microtubules / tubulin / single isoform / recombinant / dynamic instability
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Gap junction assembly / Anchoring of the basal body to the plasma membrane / 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 ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Gap junction assembly / Anchoring of the basal body to the plasma membrane / 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 / Prefoldin mediated transfer of substrate to CCT/TriC / Formation of tubulin folding intermediates by CCT/TriC / Post-chaperonin tubulin folding pathway / Recycling pathway of L1 / Hedgehog 'off' state / Cilium Assembly / COPI-dependent Golgi-to-ER retrograde traffic / Intraflagellar transport / Kinesins / RHO GTPases activate IQGAPs / Activation of AMPK downstream of NMDARs / RHO GTPases Activate Formins / Assembly and cell surface presentation of NMDA receptors / 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-mediated anterograde transport / netrin receptor binding / dorsal root ganglion development / cytoskeleton-dependent intracellular transport / regulation of synapse organization / cytoplasmic microtubule / microtubule-based process / ciliary basal body-plasma membrane docking / filopodium / cytoplasmic ribonucleoprotein granule / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron differentiation / microtubule cytoskeleton / recycling endosome / neuromuscular junction / growth cone / regulation of G2/M transition of mitotic cell cycle / axon guidance / lamellipodium / mitotic cell cycle / myelin sheath / G2/M transition of mitotic cell cycle / microtubule / GTPase activity / cell division / membrane raft / axon / GTP binding / dendrite / protein domain specific binding / structural molecule activity / extracellular exosome / nucleus / cytosol / cytoplasm
Tubulin/FtsZ, 2-layer sandwich domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin, C-terminal / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily ...Tubulin/FtsZ, 2-layer sandwich domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin, C-terminal / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ family, GTPase domain / Tubulin C-terminal domain / Tubulin subunits alpha, beta, and gamma signature. / Tubulin-beta mRNA autoregulation signal. / Tubulin
Tubulin beta-3 chain / Tubulin alpha-1A chain
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsVemu, A. / Atherton, J. / Spector, J.O. / Szyk, A. / Moores, C.A. / Roll-Mecak, A.
Funding support United Kingdom, United States, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
National Institutes of Health/National Institute of Neurological Disorders and Stroke United States
National Institutes of Health/National Heart, Lung, and Blood Institute 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 15, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 4, 2016Provider: repository / Type: Initial release
Revision 1.1May 11, 2016Group: Database references
Revision 1.2Jun 29, 2016Group: Database references
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Data collection / Database references
Category: citation / em_image_scans / pdbx_audit_support
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
K: Tubulin beta-3 chain
H: Tubulin alpha-1A chain
I: Tubulin beta-3 chain
E: Tubulin alpha-1A chain
J: Tubulin beta-3 chain
C: Tubulin beta-3 chain
D: Tubulin beta-3 chain
F: Tubulin alpha-1A chain
A: Tubulin alpha-1A chain
B: Tubulin alpha-1A chain
L: Tubulin beta-3 chain
G: Tubulin alpha-1A chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)585,31236
Polymers578,75412
Non-polymers6,55824
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area51040 Å2
ΔGint-309 kcal/mol
Surface area169890 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11K
21I
12K
22J
13K
23C
14K
24D
15K
25L
16H
26E
17H
27F
18H
28A
19H
29B
110H
210G
111I
211J
112I
212C
113I
213D
114I
214L
115E
215F
116E
216A
117E
217B
118E
218G
119J
219C
120J
220D
121J
221L
122C
222D
123C
223L
124D
224L
125F
225A
126F
226B
127F
227G
128A
228B
129A
229G
130B
230G

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDAuth asym-IDAuth seq-ID
11K1 - 426
21I1 - 426
12K1 - 426
22J1 - 426
13K1 - 426
23C1 - 426
14K1 - 426
24D1 - 426
15K1 - 426
25L1 - 426
16H1 - 437
26E1 - 437
17H1 - 437
27F1 - 437
18H1 - 437
28A1 - 437
19H1 - 437
29B1 - 437
110H1 - 437
210G1 - 437
111I1 - 426
211J1 - 426
112I1 - 426
212C1 - 426
113I1 - 426
213D1 - 426
114I1 - 426
214L1 - 426
115E1 - 437
215F1 - 437
116E1 - 437
216A1 - 437
117E1 - 437
217B1 - 437
118E1 - 437
218G1 - 437
119J1 - 426
219C1 - 426
120J1 - 426
220D1 - 426
121J1 - 426
221L1 - 426
122C1 - 426
222D1 - 426
123C1 - 426
223L1 - 426
124D1 - 426
224L1 - 426
125F1 - 437
225A1 - 437
126F1 - 437
226B1 - 437
127F1 - 437
227G1 - 437
128A1 - 437
228B1 - 437
129A1 - 437
229G1 - 437
130B1 - 437
230G1 - 437

NCS ensembles:
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

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Components

#1: Protein/peptide
Tubulin beta-3 chain / Tubulin beta-4 chain / Tubulin beta-III


Mass: 47809.926 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBB3, TUBB4 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: Q13509
#2: Protein/peptide
Tubulin alpha-1A chain / Alpha-tubulin 3 / Tubulin B-alpha-1 / Tubulin alpha-3 chain


Mass: 48649.023 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBA1A, TUBA3 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: Q71U36
#3: Chemical
ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C11H18N5O13P3 / Comment: GMP-CPP (energy-carrying molecule analogue) *YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Formula: Mg / Magnesium
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
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 / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: MicrotubulesMicrotubule / Type: ORGANELLE OR CELLULAR COMPONENT
Details: 14pf GMPCPP-bound microtubule composed of human beta3 and alpha1a tubulin
Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightValue: 110 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF9 / Plasmid: pFastBac-Dual
Buffer solutionpH: 6.9
Buffer componentName: BRB80
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: 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)

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Processing

SoftwareName: REFMAC / Version: 5.8.0133 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -25.71 ° / Axial rise/subunit: 8.86 Å / Axial symmetry: C14
3D reconstructionResolution: 4 Å / Resolution method: OTHER / Num. of particles: 10164
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.
Symmetry type: HELICAL
RefinementResolution: 4→214.9 Å / Cor.coef. Fo:Fc: 0.863 / SU B: 58.573 / SU ML: 0.666 / σ(F): 0
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3793 --
Obs0.3793 102758 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 220.7 Å2 / Biso mean: 97.212 Å2 / Biso min: 4.6 Å2
Baniso -1Baniso -2Baniso -3
1--6.47 Å2-0.02 Å2-1.2 Å2
2--0.74 Å21.64 Å2
3---5.72 Å2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0090.01941454
r_bond_other_d0.0030.0238268
r_angle_refined_deg1.4721.95756304
r_angle_other_deg1.048387996
r_dihedral_angle_1_deg5.71355106
r_dihedral_angle_2_deg32.26424.0981962
r_dihedral_angle_3_deg16.143156732
r_dihedral_angle_4_deg15.27115252
r_chiral_restr0.0870.26162
r_gen_planes_refined0.0060.02147226
r_gen_planes_other0.0020.029798
r_mcbond_it4.5959.49520478
r_mcbond_other4.5959.49420477
r_mcangle_it8.10714.23825566
Refine LS restraints NCS

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

Ens-IDDom-IDAuth asym-IDNumber
11K50726
12I50726
21K50720
22J50720
31K50720
32C50720
41K50724
42D50724
51K50728
52L50728
61H51368
62E51368
71H51362
72F51362
81H51366
82A51366
91H51366
92B51366
101H51360
102G51360
111I50716
112J50716
121I50722
122C50722
131I50720
132D50720
141I50722
142L50722
151E51364
152F51364
161E51374
162A51374
171E51366
172B51366
181E51360
182G51360
191J50716
192C50716
201J50720
202D50720
211J50722
212L50722
221C50716
222D50716
231C50722
232L50722
241D50720
242L50720
251F51366
252A51366
261F51366
262B51366
271F51358
272G51358
281A51368
282B51368
291A51360
292G51360
301B51360
302G51360
LS refinement shellResolution: 4→4.104 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.543 7593 -
Obs--100 %

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