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- EMDB-8058: Structural basis of backwards motion in kinesin-14: minus-end dir... -

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Basic information

Entry
Database: EMDB / ID: EMD-8058
TitleStructural basis of backwards motion in kinesin-14: minus-end directed nKn664 in the AMPPNP state
Map dataNone
Sample
  • Complex: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubule
    • Protein or peptide: Minus-end kinesin-1/kinesin-14 chimera nKn664
Function / homology
Function and homology information


minus-end directed microtubule sliding / distributive segregation / regulation of mitotic spindle elongation / meiotic spindle assembly / mitotic spindle elongation / mitotic spindle microtubule / meiotic spindle organization / spindle assembly involved in female meiosis / minus-end-directed microtubule motor activity / regulation of mitotic spindle assembly ...minus-end directed microtubule sliding / distributive segregation / regulation of mitotic spindle elongation / meiotic spindle assembly / mitotic spindle elongation / mitotic spindle microtubule / meiotic spindle organization / spindle assembly involved in female meiosis / minus-end-directed microtubule motor activity / regulation of mitotic spindle assembly / microtubule bundle formation / meiotic spindle / mitotic centrosome separation / positive regulation of axon guidance / spindle organization / mitotic spindle assembly / mRNA transport / cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / mitotic spindle organization / chromosome segregation / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / double-stranded RNA binding / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / centrosome / ubiquitin protein ligase binding / GTP binding / protein homodimerization activity / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin ...Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein claret segregational / Tubulin alpha-1B chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsShigematsu H / Yokoyama T / Kikkawa M / Shirouzu M / Nitta R
CitationJournal: Structure / Year: 2016
Title: Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.
Authors: Masahiko Yamagishi / Hideki Shigematsu / Takeshi Yokoyama / Masahide Kikkawa / Mitsuhiro Sugawa / Mari Aoki / Mikako Shirouzu / Junichiro Yajima / Ryo Nitta /
Abstract: Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is ...Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.
History
DepositionJan 18, 2016-
Header (metadata) releaseSep 7, 2016-
Map releaseSep 7, 2016-
UpdateDec 7, 2016-
Current statusDec 7, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5hnw
  • Surface level: 3.9
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5hnw
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8058.png

Downloads & links

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Map

FileDownload / File: emd_8058.map.gz / Format: CCP4 / Size: 12 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.284 Å
Density
Contour LevelBy AUTHOR: 3.9 / Movie #1: 3.9
Minimum - Maximum-5.714083 - 16.014668
Average (Standard dev.)0.9187484 (±2.9597764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin256256384
Dimensions128128192
Spacing128128192
CellA: 164.352 Å / B: 164.352 Å / C: 246.52802 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2841.2841.284
M x/y/z128128192
origin x/y/z0.0000.0000.000
length x/y/z164.352164.352246.528
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ129141209
MAP C/R/S123
start NC/NR/NS256256384
NC/NR/NS128128192
D min/max/mean-5.71416.0150.919

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Supplemental data

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Sample components

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Entire : Minus-end directed Ncd chimera nKn664 in the AMPPNP state complex...

EntireName: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubule
Components
  • Complex: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubule
    • Protein or peptide: Minus-end kinesin-1/kinesin-14 chimera nKn664

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Supramolecule #1: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complex...

SupramoleculeName: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubule
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: Minus-end kinesin-1/kinesin-14 chimera nKn664

MacromoleculeName: Minus-end kinesin-1/kinesin-14 chimera nKn664 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 41.857551 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KEQLFQSNME RKELHNTVMD LRGNIKVMCR FRPLNEAEIL RGDKFIPKFK GEETVVI QG KPYVFDRVLP PNTTQEQVYN ACAKQIVKDV LEGYNGTIFA YGQTSSGKT HTMEGKLHDP QLMGIIPRIA HDIFDHIYSM DENLEFA IK VSYFEIYLDK IRDLLDVSKT ...String:
KEQLFQSNME RKELHNTVMD LRGNIKVMCR FRPLNEAEIL RGDKFIPKFK GEETVVI QG KPYVFDRVLP PNTTQEQVYN ACAKQIVKDV LEGYNGTIFA YGQTSSGKT HTMEGKLHDP QLMGIIPRIA HDIFDHIYSM DENLEFA IK VSYFEIYLDK IRDLLDVSKT NLAVHEDKNR VPYVKGCTER FVSSPEEVMD VI DEGKSNR HVAVTNMNEH SSRSHSIFLI NIKQENVETE KKLSGKLYLV DLAGSEKVSK TGAEGAVLDE K NINKSLSA LGNVISALAE GTTHVPYRDS KM TRILQDS LGGNCRTTIV ICCSPSVFNE AETKSTLMFA ASVNSCKMTK AKRNRYLNNS VANSSTQSNN SGSFDK

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.648646 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.719666 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Details: High-resolution noise substitution was performed. / Number images used: 229516

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-5hnw:
Structural basis of backwards motion in kinesin-14: minus-end directed nKn664 in the AMPPNP state

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