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- PDB-6dpv: Undecorated GDP microtubule -

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Basic information

Entry
Database: PDB / ID: 6dpv
TitleUndecorated GDP microtubule
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsCELL CYCLE / microtubule / cytoskeleton / dynamic / seam
Function / homology
Function and homology information


RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic ...RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / RHO GTPases Activate Formins / microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule / GTPase activity / GTP binding / cytoplasm
Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin ...Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site
Tubulin beta chain / Tubulin alpha-1B chain
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhang, R. / Nogales, E.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM051487 United States
National Science Foundation (United States)1106400 United States
Howard Hughes Medical Institute United States
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018
Title: Separating the effects of nucleotide and EB binding on microtubule structure.
Authors: Rui Zhang / Benjamin LaFrance / Eva Nogales /
Abstract: Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and ...Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and are highly regulated by a number of MT-associated proteins (MAPs). How MAPs affect MTs is still not fully understood, partly due to a lack of high-resolution structural data on undecorated MTs, which need to serve as a baseline for further comparisons. Here we report three structures of MTs in different nucleotide states (GMPCPP, GDP, and GTPγS) at near-atomic resolution and in the absence of any binding proteins. These structures allowed us to differentiate the effects of nucleotide state versus MAP binding on MT structure. Kinesin binding has a small effect on the extended, GMPCPP-bound lattice, but hardly affects the compacted GDP-MT lattice, while binding of end-binding (EB) proteins can induce lattice compaction (together with lattice twist) in MTs that were initially in an extended and more stable state. We propose a MT lattice-centric model in which the MT lattice serves as a platform that integrates internal tubulin signals, such as nucleotide state, with outside signals, such as binding of MAPs or mechanical forces, resulting in global lattice rearrangements that in turn affect the affinity of other MT partners and result in the exquisite regulation of MT dynamics.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 9, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)606,61730
Polymers600,67312
Non-polymers5,94418
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area49620 Å2
ΔGint-270 kcal/mol
Surface area168620 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21C
12A
22E
13A
23J
14A
24K
15A
25L
16B
26D
17B
27F
18B
28G
19B
29H
110B
210I
111C
211E
112C
212J
113C
213K
114C
214L
115D
215F
116D
216G
117D
217H
118D
218I
119E
219J
120E
220K
121E
221L
122F
222G
123F
223H
124F
224I
125G
225H
126G
226I
127H
227I
128J
228K
129J
229L
130K
230L

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDAuth asym-IDAuth seq-ID
11A1 - 437
21C1 - 437
12A1 - 437
22E1 - 437
13A1 - 437
23J1 - 437
14A1 - 437
24K1 - 437
15A1 - 437
25L1 - 437
16B1 - 426
26D1 - 426
17B1 - 426
27F1 - 426
18B1 - 426
28G1 - 426
19B1 - 426
29H1 - 426
110B1 - 426
210I1 - 426
111C1 - 437
211E1 - 437
112C1 - 437
212J1 - 437
113C1 - 437
213K1 - 437
114C1 - 437
214L1 - 437
115D1 - 426
215F1 - 426
116D1 - 426
216G1 - 426
117D1 - 426
217H1 - 426
118D1 - 426
218I1 - 426
119E1 - 437
219J1 - 437
120E1 - 437
220K1 - 437
121E1 - 437
221L1 - 437
122F1 - 426
222G1 - 426
123F1 - 426
223H1 - 426
124F1 - 426
224I1 - 426
125G1 - 426
225H1 - 426
126G1 - 426
226I1 - 426
127H1 - 426
227I1 - 426
128J1 - 437
228K1 - 437
129J1 - 437
229L1 - 437
130K1 - 437
230L1 - 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 alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q2XVP4
#2: Protein/peptide
Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Dynamic microtubule in GDP state / Type: COMPLEX / Entity ID: 1, 2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Buffer solutionpH: 6.8
Details: BRB80 buffer (80 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgCl2) supplemented with 1 mM GTP, 1 mM DTT, and 0.05% Nonident P-40
SpecimenConc.: 3 mg/ml
Details: Microtubules were polymerized at 37 degrees for 30 minutes.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was glow discharged by Solarus (Gatan Inc).
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 310 K / Details: Blot for 4 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2700 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 1.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 695
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

SoftwareName: REFMAC / Version: 5.8.0091 / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9EMANinitial Euler assignment
10FREALIGNfinal Euler assignment
12FREALIGN3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -25.766 ° / Axial rise/subunit: 8.754 Å / Axial symmetry: C1
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48097 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 150 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
RefinementResolution: 3.3→211.48 Å / Cor.coef. Fo:Fc: 0.743 / SU B: 37.519 / SU ML: 0.596 / ESU R: 1.857
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.41195 --
Obs0.41195 183733 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 32.104 Å2
Baniso -1Baniso -2Baniso -3
1--2.36 Å2-0.37 Å20.12 Å2
2--5.7 Å20.07 Å2
3----3.34 Å2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0080.01941436
r_bond_other_d0.0020.0238172
r_angle_refined_deg1.3831.95856304
r_angle_other_deg0.951387774
r_dihedral_angle_1_deg11.0044.8245766
r_dihedral_angle_2_deg35.57324.1641967
r_dihedral_angle_3_deg15.896156652
r_dihedral_angle_4_deg13.05815257
r_chiral_restr0.0790.26156
r_gen_planes_refined0.0050.02147376
r_gen_planes_other0.0020.029798
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_it1.1043.18420478
r_mcbond_other1.1043.18420477
r_mcangle_it2.0594.77425566
r_mcangle_other2.0594.77425567
r_scbond_it0.8463.29820958
r_scbond_other0.8463.320946
r_scangle_it
r_scangle_other1.644.87430719
r_long_range_B_refined4.91127.93594248
r_long_range_B_other4.91127.9494230
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
Refine LS restraints NCS

Refinement-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A519280
12C519280
21A519300
22E519300
31A518400.01
32J518400.01
41A518420.01
42K518420.01
51A519200
52L519200
61B518300
62D518300
71B518300
72F518300
81B518280
82G518280
91B518280
92H518280
101B518260
102I518260
111C519280
112E519280
121C518400.01
122J518400.01
131C518400.01
132K518400.01
141C519200
142L519200
151D518300
152F518300
161D518280
162G518280
171D518280
172H518280
181D518260
182I518260
191E518400.01
192J518400.01
201E518400.01
202K518400.01
211E519200
212L519200
221F518280
222G518280
231F518280
232H518280
241F518260
242I518260
251G518280
252H518280
261G518260
262I518260
271H518260
272I518260
281J519260
282K519260
291J518400.01
292L518400.01
301K518400.01
302L518400.01
LS refinement shellResolution: 3.3→3.386 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.749 13601 -
Rfree-0 -
Obs--100 %

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