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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


netrin receptor binding / Post-chaperonin tubulin folding pathway / axonemal microtubule / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / organelle transport along microtubule / glial cell differentiation / cytoskeleton-dependent intracellular transport ...netrin receptor binding / Post-chaperonin tubulin folding pathway / axonemal microtubule / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / organelle transport along microtubule / glial cell differentiation / cytoskeleton-dependent intracellular transport / forebrain morphogenesis / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / neuron projection arborization / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / cerebellar cortex morphogenesis / dentate gyrus development / COPI-independent Golgi-to-ER retrograde traffic / pyramidal neuron differentiation / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / centrosome cycle / COPI-dependent Golgi-to-ER retrograde traffic / motor behavior / response to L-glutamate / smoothened signaling pathway / regulation of synapse organization / intercellular bridge / locomotory exploration behavior / startle response / microtubule polymerization / Recycling pathway of L1 / RHO GTPases activate IQGAPs / Hedgehog 'off' state / response to tumor necrosis factor / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / response to mechanical stimulus / Mitotic Prometaphase / homeostasis of number of cells within a tissue / EML4 and NUDC in mitotic spindle formation / condensed chromosome / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / cellular response to calcium ion / MHC class II antigen presentation / adult locomotory behavior / AURKA Activation by TPX2 / filopodium / cell periphery / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / axon guidance / synapse organization / peptide binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / intracellular protein transport / neuron migration / neuromuscular junction / visual learning / PKR-mediated signaling / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / mitotic spindle / cerebral cortex development / memory / microtubule cytoskeleton organization / recycling endosome / Aggrephagy / HCMV Early Events / 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 / mitotic cell cycle / lamellipodium / gene expression / growth cone / neuron apoptotic process / microtubule / hydrolase activity / protein heterodimerization activity / cell division / axon / GTPase activity / dendrite / neuronal cell body / protein-containing complex binding / structural molecule activity / GTP binding / extracellular exosome / identical protein binding
Similarity search - Function
Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta-3 chain / Tubulin alpha-1A chain
Similarity search - Component
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 (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 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
Revision 1.4Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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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


  • Idetical with deposited unit
  • defined by author
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 / Beg auth comp-ID: MET / Beg label comp-ID: MET / Refine code: 0

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11GLNGLNKA1 - 4261 - 426
21GLNGLNIC1 - 4261 - 426
12GLNGLNKA1 - 4261 - 426
22GLNGLNJE1 - 4261 - 426
13GLNGLNKA1 - 4261 - 426
23GLNGLNCF1 - 4261 - 426
14GLNGLNKA1 - 4261 - 426
24GLNGLNDG1 - 4261 - 426
15GLNGLNKA1 - 4261 - 426
25GLNGLNLK1 - 4261 - 426
16VALVALHB1 - 4371 - 437
26VALVALED1 - 4371 - 437
17VALVALHB1 - 4371 - 437
27VALVALFH1 - 4371 - 437
18VALVALHB1 - 4371 - 437
28VALVALAI1 - 4371 - 437
19VALVALHB1 - 4371 - 437
29VALVALBJ1 - 4371 - 437
110VALVALHB1 - 4371 - 437
210VALVALGL1 - 4371 - 437
111GLNGLNIC1 - 4261 - 426
211GLNGLNJE1 - 4261 - 426
112GLNGLNIC1 - 4261 - 426
212GLNGLNCF1 - 4261 - 426
113GLNGLNIC1 - 4261 - 426
213GLNGLNDG1 - 4261 - 426
114GLNGLNIC1 - 4261 - 426
214GLNGLNLK1 - 4261 - 426
115VALVALED1 - 4371 - 437
215VALVALFH1 - 4371 - 437
116VALVALED1 - 4371 - 437
216VALVALAI1 - 4371 - 437
117VALVALED1 - 4371 - 437
217VALVALBJ1 - 4371 - 437
118VALVALED1 - 4371 - 437
218VALVALGL1 - 4371 - 437
119GLNGLNJE1 - 4261 - 426
219GLNGLNCF1 - 4261 - 426
120GLNGLNJE1 - 4261 - 426
220GLNGLNDG1 - 4261 - 426
121GLNGLNJE1 - 4261 - 426
221GLNGLNLK1 - 4261 - 426
122GLNGLNCF1 - 4261 - 426
222GLNGLNDG1 - 4261 - 426
123GLNGLNCF1 - 4261 - 426
223GLNGLNLK1 - 4261 - 426
124GLNGLNDG1 - 4261 - 426
224GLNGLNLK1 - 4261 - 426
125VALVALFH1 - 4371 - 437
225VALVALAI1 - 4371 - 437
126VALVALFH1 - 4371 - 437
226VALVALBJ1 - 4371 - 437
127VALVALFH1 - 4371 - 437
227VALVALGL1 - 4371 - 437
128VALVALAI1 - 4371 - 437
228VALVALBJ1 - 4371 - 437
129VALVALAI1 - 4371 - 437
229VALVALGL1 - 4371 - 437
130VALVALBJ1 - 4371 - 437
230VALVALGL1 - 4371 - 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
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
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
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C10H16N5O14P3 / 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
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01941454
ELECTRON MICROSCOPYr_bond_other_d0.0030.0238268
ELECTRON MICROSCOPYr_angle_refined_deg1.4721.95756304
ELECTRON MICROSCOPYr_angle_other_deg1.048387996
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.71355106
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.26424.0981962
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.143156732
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.27115252
ELECTRON MICROSCOPYr_chiral_restr0.0870.26162
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02147226
ELECTRON MICROSCOPYr_gen_planes_other0.0020.029798
ELECTRON MICROSCOPYr_mcbond_it4.5959.49520478
ELECTRON MICROSCOPYr_mcbond_other4.5959.49420477
ELECTRON MICROSCOPYr_mcangle_it8.10714.23825566
Refine LS restraints NCS

Refine-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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