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- EMDB-6353: Cryo-EM structure of dynamic GDP-microtubule (14 protofilaments) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6353
TitleCryo-EM structure of dynamic GDP-microtubule (14 protofilaments) decorated with kinesin
Map dataReconstruction of kinesin-bound dynamic GDP microtubule (14 protofilaments) with pseudo-helical symmetry applied
Sample
  • Sample: kinesin-bound dynamic GDP microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: kinesin
Keywordsmicrotubule / GDP / kinesin
Function / homology
Function and homology information


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 ...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 / COPI-mediated anterograde transport / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
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 ...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
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
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  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jas
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jas
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6353.gif

Downloads & links

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Map

FileDownload / File: emd_6353.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of kinesin-bound dynamic GDP microtubule (14 protofilaments) 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-12.340007780000001 - 23.404703139999999
Average (Standard dev.)0.02849031 (±0.91781855)
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-12.34023.4050.028

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Supplemental data

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Sample components

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Entire : kinesin-bound dynamic GDP microtubule

EntireName: kinesin-bound dynamic GDP microtubule
Components
  • Sample: kinesin-bound dynamic GDP microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: kinesin

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Supramolecule #1000: kinesin-bound dynamic GDP microtubule

SupramoleculeName: kinesin-bound dynamic GDP microtubule / type: sample / ID: 1000 / Oligomeric state: helical assembly / Number unique components: 3

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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: kinesin

MacromoleculeName: kinesin / type: protein_or_peptide / ID: 3
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

Concentration3 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 7, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 327 / 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: 3.0 µm / Nominal defocus min: 1.0 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: 8.75 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.76 °
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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