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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 / dorsal root ganglion development / cytoskeleton-dependent intracellular transport / regulation of synapse organization / cytoplasmic microtubule / microtubule-based process / ciliary basal body-plasma membrane docking / filopodium / neuron differentiation / structural constituent of cytoskeleton ...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 / neuron differentiation / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / microtubule cytoskeleton / neuromuscular junction / recycling endosome / microtubule cytoskeleton organization / regulation of G2/M transition of mitotic cell cycle / growth cone / axon guidance / lamellipodium / G2/M transition of mitotic cell cycle / myelin sheath / mitotic cell cycle / microtubule / GTPase activity / cell division / membrane raft / axon / protein domain specific binding / GTP binding / dendrite / structural molecule activity / extracellular exosome / identical protein binding / nucleus / cytosol / cytoplasm
Alpha tubulin / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Beta tubulin / Beta tubulin, autoregulation binding site / Tubulin, C-terminal / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Tubulin, conserved site / Tubulin/FtsZ, C-terminal domain superfamily ...Alpha tubulin / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Beta tubulin / Beta tubulin, autoregulation binding site / Tubulin, C-terminal / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Tubulin, conserved site / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Tubulin alpha-1A chain / Tubulin beta-3 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 (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.
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
Revision 1.4Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _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:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010K1 - 426
2010I1 - 426
1020K1 - 426
2020J1 - 426
1030K1 - 426
2030C1 - 426
1040K1 - 426
2040D1 - 426
1050K1 - 426
2050L1 - 426
1060H1 - 437
2060E1 - 437
1070H1 - 437
2070F1 - 437
1080H1 - 437
2080A1 - 437
1090H1 - 437
2090B1 - 437
10100H1 - 437
20100G1 - 437
10110I1 - 426
20110J1 - 426
10120I1 - 426
20120C1 - 426
10130I1 - 426
20130D1 - 426
10140I1 - 426
20140L1 - 426
10150E1 - 437
20150F1 - 437
10160E1 - 437
20160A1 - 437
10170E1 - 437
20170B1 - 437
10180E1 - 437
20180G1 - 437
10190J1 - 426
20190C1 - 426
10200J1 - 426
20200D1 - 426
10210J1 - 426
20210L1 - 426
10220C1 - 426
20220D1 - 426
10230C1 - 426
20230L1 - 426
10240D1 - 426
20240L1 - 426
10250F1 - 437
20250A1 - 437
10260F1 - 437
20260B1 - 437
10270F1 - 437
20270G1 - 437
10280A1 - 437
20280B1 - 437
10290A1 - 437
20290G1 - 437
10300B1 - 437
20300G1 - 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 / Magnesium


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
Refinement-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

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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