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- PDB-3jas: Cryo-EM structure of dynamic GDP-microtubule (14 protofilaments) ... -

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Basic information

Entry
Database: PDB / ID: 3jas
TitleCryo-EM structure of dynamic GDP-microtubule (14 protofilaments) decorated with kinesin
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / microtubule / GDP / kinesin
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: 3.5 Å
AuthorsZhang, R. / Nogales, E.
CitationJournal: Cell / Year: 2015
Title: Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins.
Authors: Rui Zhang / Gregory M Alushin / Alan Brown / Eva Nogales /
Abstract: Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six ...Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 19, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name

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Assembly

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


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


TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 8.75 Å / Rotation per n subunits: -25.76 °)
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11E
21J
12E
22C
13E
23L
14E
24A
15E
25K
16F
26G
17F
27D
18F
28I
19F
29B
110F
210H
111J
211C
112J
212L
113J
213A
114J
214K
115G
215D
116G
216I
117G
217B
118G
218H
119C
219L
120C
220A
121C
221K
122D
222I
123D
223B
124D
224H
125L
225A
126L
226K
127I
227B
128I
228H
129A
229K
130B
230H

NCS domain segments:

Component-ID: 0 / Refine code: 0

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

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
Detailspseudo-helical symmetry

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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) / Organ: brain / 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) / Organ: brain / 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
Compound detailsTHOUGH PRESENT IN THE SAMPLE, KINESIN HAS NOT BEEN MODELED.

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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

Component
IDNameTypeDetailsParent-ID
1kinesin-bound dynamic GDP microtubuleCOMPLEXhelical assembly0
2Alpha tubulin1
3Beta tubulin1
4kinesin1
Buffer solutionName: BRB80 / pH: 6.8
Details: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh C-flat 1.2/1.3 EM grid, glow discharged in Ar/O2 gas (Solarus, Gatan Inc)
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 90.4 K / Humidity: 95 %
Details: Blot once for 4 seconds before plunging into liquid ethane (FEI VITROBOT MARK II).
Method: Blot once for 4 seconds before plunging.

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Electron microscopy imaging

MicroscopyModel: FEI TITAN / Date: May 7, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Calibrated magnification: 27500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 27,500 times magnification.
Specimen holderModel: GATAN LIQUID NITROGEN / Specimen holder type: Gatan 626 holder / Temperature: 90 K
Image recordingElectron dose: 27.6 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Details: The camera was operated in counting mode with a dose rate of ~8 electrons/pixel/s on the camera. A total exposure time of 6 seconds was fractionated into 20 movie frames.
Image scansNum. digital images: 327

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0091refinement
PDB_EXTRACT3.15data extraction
EM software

Category: 3D reconstruction

IDNameVersion
1EMAN1
2FREALIGN
CTF correctionDetails: CTFFIND4, each particle
Helical symmertyAngular rotation/subunit: 25.76 ° / Axial rise/subunit: 8.75 Å / Axial symmetry: C1
3D reconstructionMethod: projection matching / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Nominal pixel size: 1.32 Å / Actual pixel size: 1.32 Å
Details: IHRSR algorithm with microtubule-specific pseudo-helical symmetry applied
Symmetry type: HELICAL
RefinementResolution: 3.5→211.2 Å / Cor.coef. Fo:Fc: 0.841 / SU B: 42.965 / SU ML: 0.644 / σ(F): 0
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3834 --
Obs0.3834 151914 100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso max: 143.07 Å2 / Biso mean: 47.605 Å2 / Biso min: 9.86 Å2
Baniso -1Baniso -2Baniso -3
1--2.97 Å2-0.03 Å20.16 Å2
2--8.14 Å2-0.49 Å2
3----5.17 Å2
Refinement stepCycle: LAST / Resolution: 3.5→211.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms40140 0 366 0 40506
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_deg1.3871.95856304
r_angle_other_deg0.959387774
r_dihedral_angle_1_deg11.8834.8245795
r_dihedral_angle_2_deg34.63124.1721963
r_dihedral_angle_3_deg15.791156655
r_dihedral_angle_4_deg12.19715253
r_chiral_restr0.0810.26156
r_gen_planes_refined0.0050.02147376
r_gen_planes_other0.0020.029798
r_mcbond_it1.7744.68320478
r_mcbond_other1.7744.68320477
r_mcangle_it3.2677.02125566
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
11E52034
12J52034
21E52032
22C52032
31E52036
32L52036
41E52034
42A52034
51E52036
52K52036
61F51868
62G51868
71F51874
72D51874
81F51870
82I51870
91F51872
92B51872
101F51868
102H51868
111J52028
112C52028
121J52038
122L52038
131J52030
132A52030
141J52038
142K52038
151G51872
152D51872
161G51874
162I51874
171G51872
172B51872
181G51874
182H51874
191C52034
192L52034
201C52030
202A52030
211C52032
212K52032
221D51876
222I51876
231D51878
232B51878
241D51874
242H51874
251L52034
252A52034
261L52044
262K52044
271I51876
272B51876
281I51876
282H51876
291A52034
292K52034
301B51874
302H51874
LS refinement shellResolution: 3.5→3.591 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.765 11219 -
Obs--100 %

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