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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


cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / structural constituent of cytoskeleton / neuron migration / microtubule cytoskeleton organization / double-stranded RNA binding / mitotic cell cycle / microtubule / GTPase activity ...cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / structural constituent of cytoskeleton / neuron migration / microtubule cytoskeleton organization / double-stranded RNA binding / mitotic cell cycle / microtubule / GTPase activity / GTP binding / ubiquitin protein ligase binding / cytoplasm
Tubulin, conserved site / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Beta tubulin / Alpha tubulin / Tubulin ...Tubulin, conserved site / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Beta tubulin / Alpha tubulin / Tubulin / Tubulin/FtsZ family, GTPase domain / Tubulin, C-terminal / Tubulin C-terminal domain / 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 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 cryo- ...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
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Structure viewerMolecule:
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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:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010E1 - 437
2010J1 - 437
1020E1 - 437
2020C1 - 437
1030E1 - 437
2030L1 - 437
1040E1 - 437
2040A1 - 437
1050E1 - 437
2050K1 - 437
1060F1 - 426
2060G1 - 426
1070F1 - 426
2070D1 - 426
1080F1 - 426
2080I1 - 426
1090F1 - 426
2090B1 - 426
10100F1 - 426
20100H1 - 426
10110J1 - 437
20110C1 - 437
10120J1 - 437
20120L1 - 437
10130J1 - 437
20130A1 - 437
10140J1 - 437
20140K1 - 437
10150G1 - 426
20150D1 - 426
10160G1 - 426
20160I1 - 426
10170G1 - 426
20170B1 - 426
10180G1 - 426
20180H1 - 426
10190C1 - 437
20190L1 - 437
10200C1 - 437
20200A1 - 437
10210C1 - 437
20210K1 - 437
10220D1 - 426
20220I1 - 426
10230D1 - 426
20230B1 - 426
10240D1 - 426
20240H1 - 426
10250L1 - 437
20250A1 - 437
10260L1 - 437
20260K1 - 437
10270I1 - 426
20270B1 - 426
10280I1 - 426
20280H1 - 426
10290A1 - 437
20290K1 - 437
10300B1 - 426
20300H1 - 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
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
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 / Guanosine triphosphate


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


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


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / 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
IDNameVersionCategory
1EMAN13D reconstruction
2FREALIGN3D reconstruction
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-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01941436
ELECTRON MICROSCOPYr_bond_other_d0.0020.0238172
ELECTRON MICROSCOPYr_angle_refined_deg1.3871.95856304
ELECTRON MICROSCOPYr_angle_other_deg0.959387774
ELECTRON MICROSCOPYr_dihedral_angle_1_deg11.8834.8245795
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.63124.1721963
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.791156655
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.19715253
ELECTRON MICROSCOPYr_chiral_restr0.0810.26156
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02147376
ELECTRON MICROSCOPYr_gen_planes_other0.0020.029798
ELECTRON MICROSCOPYr_mcbond_it1.7744.68320478
ELECTRON MICROSCOPYr_mcbond_other1.7744.68320477
ELECTRON MICROSCOPYr_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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