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- EMDB-8059: Structural basis of backwards motion in kinesin-14: minus-end dir... -

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Basic information

Entry
Database: EMDB / ID: EMD-8059
TitleStructural basis of backwards motion in kinesin-14: minus-end directed nKn664 in the nucleotide-free state
Map dataNone
Sample
  • Complex: Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubule
    • Complex: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin alpha-1B chain
    • Complex: Tubulin beta-2B chain
      • Protein or peptide: Tubulin beta-2B chain
    • Complex: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
      • Protein or peptide: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL
Keywordskinesin / kinesin-14 / microtubule / ATPase / STRUCTURAL PROTEIN-MOTOR PROTEIN complex
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 / microtubule bundle formation / spindle assembly involved in female meiosis / 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 / microtubule bundle formation / spindle assembly involved in female meiosis / regulation of mitotic spindle assembly / mitotic centrosome separation / positive regulation of axon guidance / meiotic spindle / minus-end-directed microtubule motor activity / spindle organization / mitotic spindle assembly / mRNA transport / microtubule-based process / cytoplasmic microtubule / cellular response to interleukin-4 / mitotic spindle organization / chromosome segregation / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / microtubule cytoskeleton organization / spindle / neuron migration / mitotic cell cycle / double-stranded RNA binding / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / cilium / protein heterodimerization activity / cell division / GTPase activity / ubiquitin protein ligase binding / centrosome / GTP binding / protein homodimerization activity / ATP binding / metal ion binding / nucleus / cytoplasm / cytosol
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 / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...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 / 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 / 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 speciesBos taurus (domestic cattle) / Drosophila melanogaster (fruit fly)
Methodhelical reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsShigematsu H / Yokoyama T
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
DepositionApr 5, 2016-
Header (metadata) releaseJun 22, 2016-
Map releaseAug 10, 2016-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5hnx
  • Surface level: 3.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5hnx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8059.png

Downloads & links

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Map

FileDownload / File: emd_8059.map.gz / Format: CCP4 / Size: 8.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 192 pix.
= 246.528 Å		1.28 Å/pix.
x 108 pix.
= 138.672 Å		1.28 Å/pix.
x 108 pix.
= 138.672 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.284 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.1 / Movie #1: 3.1
Minimum - Maximum-7.321161 - 18.355370000000001
Average (Standard dev.)1.1209195 (±3.3602126)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin276276384
Dimensions108108192
Spacing108108192
CellA: 138.672 Å / B: 138.672 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/z108108192
origin x/y/z0.0000.0000.000
length x/y/z138.672138.672246.528
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ129141209
MAP C/R/S123
start NC/NR/NS276276384
NC/NR/NS108108192
D min/max/mean-7.32118.3551.121

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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
    • Complex: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin alpha-1B chain
    • Complex: Tubulin beta-2B chain
      • Protein or peptide: Tubulin beta-2B chain
    • Complex: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
      • Protein or peptide: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL

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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: #1-#3

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Supramolecule #2: Tubulin alpha-1B chain

SupramoleculeName: Tubulin alpha-1B chain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (domestic cattle)

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Supramolecule #3: Tubulin beta-2B chain

SupramoleculeName: Tubulin beta-2B chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (domestic cattle)

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Supramolecule #4: Protein claret segregational,kinesin-1/kinesin-14,Protein claret ...

SupramoleculeName: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 50.107238 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRGHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEEGEDEA

UniProtKB: Tubulin beta-2B chain

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Macromolecule #3: Protein claret segregational,kinesin-1/kinesin-14,Protein claret ...

MacromoleculeName: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 41.62025 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KEQLFQSNME RKELHNTVMD LRGNIKVMCR FRPLNEAEIL RGDKFIPKFK GEETVVIQGK PYVFDRVLPP NTTQEQVYNA CAKQIVKDV LEGYNGTIFA YGQTSSGKTH TMEGKLHDPQ LMGIIPRIAH DIFDHIYSMD ENLEFAIKVS YFEIYLDKIR D LLDVSKTN ...String:
KEQLFQSNME RKELHNTVMD LRGNIKVMCR FRPLNEAEIL RGDKFIPKFK GEETVVIQGK PYVFDRVLPP NTTQEQVYNA CAKQIVKDV LEGYNGTIFA YGQTSSGKTH TMEGKLHDPQ LMGIIPRIAH DIFDHIYSMD ENLEFAIKVS YFEIYLDKIR D LLDVSKTN LAVHEDKNRV PYVKGCTERF VSSPEEVMDV IDEGKSNRHV AVTNMNEHSS RSHSIFLINI KQENVETEKK LS GKLYLVD LAGSEKVSKT GAEGAVLDEA KNINKSLSAL GNVISALAEG TTHVPYRDSK MTRILQDSLG GNCRTTIVIC CSP SVFNEA ETKSTLMFAA SVNSCKMTKA KRNRYLNNSV ANSSTQSNNS GSFDK

UniProtKB: Protein claret segregational, Protein claret segregational

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #7: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 7 / Number of copies: 1 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication*YM

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

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 8.751208 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.725189 °
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
Startup modelType of model: EMDB MAP
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-5hnx:
Structural basis of backwards motion in kinesin-14: minus-end directed nKn664 in the nucleotide-free state

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