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- PDB-5hnx: Structural basis of backwards motion in kinesin-14: minus-end dir... -

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Basic information

Entry
Database: PDB / ID: 5hnx
TitleStructural basis of backwards motion in kinesin-14: minus-end directed nKn664 in the nucleotide-free state
Components
  • Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsSTRUCTURAL PROTEIN/MOTOR PROTEIN / kinesin / kinesin-14 / microtubule / ATPase / STRUCTURAL PROTEIN-MOTOR PROTEIN complex
Function / homologyTubulin / Kinesin-like protein / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin C-terminal domain / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin/FtsZ, GTPase domain superfamily ...Tubulin / Kinesin-like protein / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin C-terminal domain / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Kinesin motor domain profile. / Tubulin, C-terminal / Kinesin motor domain, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, conserved site / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Kinesin motor domain / Beta tubulin / Kinesin motor domain signature. / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / RHO GTPases activate IQGAPs / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / Hedgehog 'on' state / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recycling pathway of L1 / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / MHC class II antigen presentation / Alpha tubulin / spindle assembly involved in female meiosis / regulation of mitotic spindle elongation / spindle assembly involved in meiosis / minus-end directed microtubule sliding / distributive segregation / mitotic spindle microtubule / mitotic spindle elongation / meiotic spindle organization / ATP-dependent microtubule motor activity, minus-end-directed / microtubule bundle formation / positive regulation of axon guidance / regulation of mitotic spindle assembly / meiotic spindle / mitotic centrosome separation / kinesin complex / mRNA transport / cytoplasmic microtubule / mitotic spindle assembly / cellular response to interleukin-4 / microtubule-based process / spindle organization / microtubule motor activity / microtubule cytoskeleton organization / microtubule-based movement / mitotic spindle organization / structural constituent of cytoskeleton / neuron migration / chromosome segregation / microtubule cytoskeleton / spindle / double-stranded RNA binding / microtubule / microtubule binding / ATPase activity / GTPase activity / centrosome / myelin sheath / cell division / GTP binding / ubiquitin protein ligase binding / protein heterodimerization activity / protein homodimerization activity / ATP binding / nucleus / cytosol / cytoplasm / Protein claret segregational / Tubulin alpha-1B chain / Tubulin beta-2B chain
Function and homology information
Specimen sourceDrosophila melanogaster (fruit fly)
Rattus norvegicus (Norway rat)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 6.6 Å resolution
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 19, 2016 / Release: Aug 10, 2016
RevisionDateData content typeGroupProviderType
1.0Aug 10, 2016Structure modelrepositoryInitial release
1.1Aug 17, 2016Structure modelDatabase references
1.2Sep 7, 2016Structure modelOther
1.3Nov 2, 2016Structure modelOther

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta-2B chain
K: Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,4807
Polyers141,6353
Non-polymers1,8454
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)9310
ΔGint (kcal/M)-49
Surface area (Å2)43320

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Components

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Protein/peptide , 3 types, 3 molecules ABK

#1: Protein/peptide Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50107.238 Da / Num. of mol.: 1 / Source: (natural) Bos taurus (cattle) / References: UniProt: P81947
#2: Protein/peptide Tubulin beta-2B chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 1 / Source: (natural) Bos taurus (cattle) / References: UniProt: Q6B856
#3: Protein/peptide Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregational


Mass: 41620.250 Da / Num. of mol.: 1 / Fragment: UNP residues 325-348,UNP residues 664-700
Source: (gene. exp.) Drosophila melanogaster (fruit fly), (gene. exp.) Rattus norvegicus (Norway rat)
Gene: ncd / Production host: Escherichia coli (E. coli) / References: UniProt: P20480

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 1 / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM
#7: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Formula: C47H51NO14 / Paclitaxel

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Minus-end directed Ncd chimera nKn664 in the AMPPNP state complexed with GDP-taxol microtubuleCOMPLEX1, 2, 30MULTIPLE SOURCES
2Tubulin alpha-1B chainCOMPLEX11NATURAL
3Tubulin beta-2B chainCOMPLEX21NATURAL
4Protein claret segregational,kinesin-1/kinesin-14,Protein claret segregationalCOMPLEX31RECOMBINANT
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129913Bos taurus (cattle)
239913Bos taurus (cattle)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismPlasmid
147227Drosophila melanogaster (fruit fly)unknown
2410116Rattus norvegicus (Norway rat)unknown
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 30 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -25.725189 deg. / Axial rise/subunit: 8.751208 Å / Axial symmetry: C1
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 229516 / Details: High-resolution noise substitution was performed / Symmetry type: HELICAL
Atomic model buildingRef protocol: FLEXIBLE FIT

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