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- EMDB-6348: Cryo-EM structure of GTPgammaS-microtubule co-polymerized with EB... -

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Basic information

Entry
Database: EMDB / ID: EMD-6348
TitleCryo-EM structure of GTPgammaS-microtubule co-polymerized with EB3 and decorated with kinesin
Map dataReconstruction of EB3 and kinesin bound GTPgammaS microtubule with pseudo-helical symmetry applied
Sample
  • Sample: EB3- and kinesin-bound GTPgammaS microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: EB3
  • Protein or peptide: kinesin
Keywordsmicrotubule / EB3 / GTPgammaS / kinesin
Function / homology
Function and homology information


mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / microtubule plus-end binding / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin ...mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / microtubule plus-end binding / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / microtubule organizing center / COPI-mediated anterograde transport / regulation of microtubule polymerization / positive regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of protein kinase activity / spindle midzone / spindle assembly / regulation of microtubule polymerization or depolymerization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / protein localization / mitotic cell cycle / midbody / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cell division / GTPase activity / GTP binding / protein kinase binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Alpha tubulin ...EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin beta chain / Tubulin alpha-1B chain / Microtubule-associated protein RP/EB family member 3
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang R / Alushin GM / Brown A / 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.
History
DepositionJun 15, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseAug 12, 2015-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6348.gif

Downloads & links

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Map

FileDownload / File: emd_6348.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of EB3 and kinesin bound GTPgammaS microtubule with pseudo-helical symmetry applied
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-11.46850967 - 20.64797592
Average (Standard dev.)0.03621041 (±0.90966207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 675.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z675.840675.840675.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-11.46920.6480.036

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Supplemental data

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Sample components

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Entire : EB3- and kinesin-bound GTPgammaS microtubule

EntireName: EB3- and kinesin-bound GTPgammaS microtubule
Components
  • Sample: EB3- and kinesin-bound GTPgammaS microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: EB3
  • Protein or peptide: kinesin

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Supramolecule #1000: EB3- and kinesin-bound GTPgammaS microtubule

SupramoleculeName: EB3- and kinesin-bound GTPgammaS microtubule / type: sample / ID: 1000
Details: GTPgammaS microtubules were polymerized on the EM grid in the presence of GMPCPP-MT seeds and excess EB3, then washed with kinesin.
Oligomeric state: helical assembly / Number unique components: 4

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Macromolecule #1: Alpha tubulin

MacromoleculeName: Alpha tubulin / type: protein_or_peptide / ID: 1
Details: Porcine tubulin powder was purchased from Cytoskeleton Inc.
Oligomeric state: Helical assembly / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Porcine / Tissue: Brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 55 KDa
SequenceUniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Beta tubulin

MacromoleculeName: Beta tubulin / type: protein_or_peptide / ID: 2
Details: Porcine tubulin powder was purchased from Cytoskeleton Inc.
Oligomeric state: Helical assembly / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Porcine / Tissue: Brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 55 KDa
SequenceUniProtKB: Tubulin beta chain

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Macromolecule #3: EB3

MacromoleculeName: EB3 / type: protein_or_peptide / ID: 3 / Name.synonym: MAPRE3
Details: A monomeric construct containing residues 1-200, which lacks the C-terminal dimerization domain
Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 23 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-CodonPlus-(DE3)-RIL / Recombinant plasmid: 2BT
SequenceUniProtKB: Microtubule-associated protein RP/EB family member 3

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Macromolecule #4: kinesin

MacromoleculeName: kinesin / type: protein_or_peptide / ID: 4
Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., ...Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., et al. (1999). A structural change in the kinesin motor protein that drives motility. Nature 402, 778-784.
Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 36 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 6.8
Details: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40
GridDetails: 400 mesh C-flat 1.2/1.3 EM grid, glow discharged in Ar/O2 gas (Solarus, Gatan Inc)
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90.4 K / Instrument: FEI VITROBOT MARK II / Method: Blot once for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 27,500 times magnification.
DetailsThe camera was operated in counting mode, with a dose rate of ~8 electrons/pixel/s on the camera. A total exposure time of 6 s was fractionated into 20 movie frames.
DateMay 16, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 227 / Average electron dose: 27.6 e/Å2
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 s was fractionated into 20 movie frames.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 27500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 27500
Sample stageSpecimen holder: Gatan 626 holder / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsIHRSR algorithm with microtubule-specific pseudo-helical symmetry applied
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.45 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.72 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: EMAN1, FREALIGN
Details: Pseudo-helical symmetry was applied during the reconstruction step.
CTF correctionDetails: CTFFIND4, each particle

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