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- PDB-3jak: Cryo-EM structure of GTPgammaS-microtubule co-polymerized with EB... -

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Entry
Database: PDB / ID: 3jak
TitleCryo-EM structure of GTPgammaS-microtubule co-polymerized with EB3 (merged dataset with and without kinesin bound)
Components
  • Microtubule-associated protein RP/EB family member 3
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / microtubule / EB3 / GTPgammaS / kinesin
Function / homology
Function and homology information


Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Intraflagellar 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 / COPI-dependent Golgi-to-ER retrograde traffic ...Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Intraflagellar 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 / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / RHO GTPases activate IQGAPs / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / mitotic spindle astral microtubule end / positive regulation of microtubule plus-end binding / protein localization to microtubule plus-end / protein localization to microtubule / microtubule plus-end / microtubule plus-end binding / regulation of microtubule polymerization / regulation of microtubule polymerization or depolymerization / spindle assembly / cytoplasmic microtubule / microtubule-based process / positive regulation of protein kinase activity / spindle midzone / positive regulation of cyclin-dependent protein serine/threonine kinase activity / protein localization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / microtubule cytoskeleton / microtubule organizing center / midbody / mitotic cell cycle / microtubule / microtubule binding / protein C-terminus binding / GTPase activity / cell division / GTP binding / protein kinase binding / positive regulation of transcription, DNA-templated / perinuclear region of cytoplasm / identical protein binding / cytoplasm
Tubulin/FtsZ, C-terminal / EB3 / Calponin homology domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / EB1, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain ...Tubulin/FtsZ, C-terminal / EB3 / Calponin homology domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / EB1, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / CH domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Intermediate filament, rod domain, coil 1B / Tubulin/FtsZ family, GTPase domain / Calponin homology (CH) domain / EB1-like C-terminal motif / Tubulin C-terminal domain / Tubulin subunits alpha, beta, and gamma signature. / Tubulin-beta mRNA autoregulation signal. / Calponin homology (CH) domain profile. / Tubulin / EB1-C terminal (EB1-C) domain profile.
Tubulin beta chain / Tubulin alpha-1B chain / Microtubule-associated protein RP/EB family member 3
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 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 16, 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
N: Microtubule-associated protein RP/EB family member 3
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
M: Microtubule-associated protein RP/EB family member 3
K: Tubulin alpha-1B chain
H: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)652,92432
Polymers646,40414
Non-polymers6,52018
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 9.45 Å / Rotation per n subunits: -27.72 °)
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
111N
211M
112J
212C
113J
213L
114J
214A
115J
215K
116G
216D
117G
217I
118G
218B
119G
219H
120C
220L
121C
221A
122C
222K
123D
223I
124D
224B
125D
225H
126L
226A
127L
227K
128I
228B
129I
229H
130A
230K
131B
231H

NCS domain segments:

Component-ID: 0 / Refine code: 0

Dom-IDEns-IDAuth asym-IDAuth seq-ID
11E1 - 441
21J1 - 441
12E1 - 441
22C1 - 441
13E1 - 441
23L1 - 441
14E1 - 441
24A1 - 441
15E1 - 441
25K1 - 441
16F1 - 429
26G1 - 429
17F1 - 429
27D1 - 429
18F1 - 429
28I1 - 429
19F1 - 429
29B1 - 429
110F1 - 429
210H1 - 429
111N1 - 131
211M1 - 131
112J1 - 441
212C1 - 441
113J1 - 441
213L1 - 441
114J1 - 441
214A1 - 441
115J1 - 441
215K1 - 441
116G1 - 429
216D1 - 429
117G1 - 429
217I1 - 429
118G1 - 429
218B1 - 429
119G1 - 429
219H1 - 429
120C1 - 441
220L1 - 441
121C1 - 441
221A1 - 441
122C1 - 441
222K1 - 441
123D1 - 429
223I1 - 429
124D1 - 429
224B1 - 429
125D1 - 429
225H1 - 429
126L1 - 441
226A1 - 441
127L1 - 441
227K1 - 441
128I1 - 429
228B1 - 429
129I1 - 429
229H1 - 429
130A1 - 441
230K1 - 441
131B1 - 429
231H1 - 429

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
31
Detailspseudo-helical symmetry

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Components

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Protein/peptide , 3 types, 14 molecules EJCLAKFGDIBHNM

#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: Protein/peptide Microtubule-associated protein RP/EB family member 3 / EB1 protein family member 3 / EBF3 / End-binding protein 3 / EB3 / RP3


Mass: 22865.203 Da / Num. of mol.: 2 / Fragment: UNP residues 1-200
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPRE3 / Plasmid: 2BT / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21-CodonPlus-(DE3)-RIL / References: UniProt: Q9UPY8

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Non-polymers , 3 types, 18 molecules

#4: 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
#5: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 539.246 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3S

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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
1EB3- and kinesin-bound GTPgammaS microtubuleCOMPLEXhelical assembly0
2Alpha tubulin1
3Beta tubulin1
4EB31
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 16, 2013
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: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 27,500 times magnification.
Camera length: 0 mm
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: 345
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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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: 27.72 ° / Axial rise/subunit: 9.45 Å / Axial symmetry: C1
3D reconstructionMethod: projection matching / Resolution: 3.3 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: 0.847 / SU B: 31.753 / SU ML: 0.499 / σ(F): 0 / ESU R: 1.837
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3678 --
Obs0.3678 193403 100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso max: 176.53 Å2 / Biso mean: 29.829 Å2 / Biso min: 7.94 Å2
Baniso -1Baniso -2Baniso -3
1--0.72 Å2-0.9 Å2-0.26 Å2
2--3.03 Å20.07 Å2
3----2.31 Å2
Refinement stepCycle: LAST / Resolution: 3.5→211.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms42676 0 390 0 43066
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0080.01944052
r_bond_other_d0.0020.0240656
r_angle_refined_deg1.3311.95859830
r_angle_other_deg0.931393530
r_dihedral_angle_1_deg11.4144.8546048
r_dihedral_angle_2_deg35.04124.2492104
r_dihedral_angle_3_deg14.078157144
r_dihedral_angle_4_deg10.89315262
r_chiral_restr0.0770.26528
r_gen_planes_refined0.0050.02150264
r_gen_planes_other0.0010.0210400
r_mcbond_it1.0062.96821692
r_mcbond_other1.0062.96821691
r_mcangle_it1.8884.44327080
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
11E52906
12J52906
21E52902
22C52902
31E52902
32L52902
41E52906
42A52906
51E52902
52K52902
61F52638
62G52638
71F52640
72D52640
81F52636
82I52636
91F52642
92B52642
101F52642
102H52642
111N15968
112M15968
121J52904
122C52904
131J52908
132L52908
141J52904
142A52904
151J52906
152K52906
161G52636
162D52636
171G52640
172I52640
181G52640
182B52640
191G52640
192H52640
201C52902
202L52902
211C52902
212A52902
221C52900
222K52900
231D52634
232I52634
241D52642
242B52642
251D52640
252H52640
261L52900
262A52900
271L52902
272K52902
281I52638
282B52638
291I52640
292H52640
301A52900
302K52900
311B52644
312H52644
LS refinement shellResolution: 3.5→3.591 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.757 14402 -
Obs--100 %

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