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- PDB-8yy3: Kinesin-14 in nucleotide-free state bound to 14 PF Microtubule -

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Basic information

Entry
Database: PDB / ID: 8yy3
TitleKinesin-14 in nucleotide-free state bound to 14 PF Microtubule
Components
  • Protein claret segregational
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsCELL CYCLE / Kinesin Motor Proteins / Force Production / Power Stroke Fluctuations / Motor Spring-like Element / Reversed Motility / Mechanochemical Coupling / Mechanical States
Function / homology
Function and homology information


minus-end directed microtubule sliding / distributive segregation / regulation of mitotic spindle elongation / meiotic spindle assembly / Regulation of PLK1 Activity at G2/M Transition / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Recruitment of mitotic centrosome proteins and complexes ...minus-end directed microtubule sliding / distributive segregation / regulation of mitotic spindle elongation / meiotic spindle assembly / Regulation of PLK1 Activity at G2/M Transition / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Recruitment of mitotic centrosome proteins and complexes / mitotic spindle elongation / odontoblast differentiation / mitotic spindle microtubule / meiotic spindle organization / Neutrophil degranulation / microtubule bundle formation / spindle assembly involved in female meiosis / regulation of mitotic spindle assembly / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / mitotic centrosome separation / meiotic spindle / 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 / minus-end-directed microtubule motor activity / spindle organization / COPI-mediated anterograde transport / regulation of synapse organization / nuclear envelope lumen / MHC class I protein binding / mitotic spindle assembly / mRNA transport / intercellular bridge / spindle assembly / 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 / cytoplasmic ribonucleoprotein granule / mitotic spindle / mitotic cell cycle / microtubule cytoskeleton / cell body / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / cell division / GTPase activity / ubiquitin protein ligase binding / centrosome / 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 / 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
ADENOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Protein claret segregational / Tubulin alpha-1B chain / Tubulin beta chain
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsShibata, S. / Imasaki, T. / Shigematsu, H. / Endow, S.A. / Nitta, R.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJFR214K Japan
Japan Science and TechnologyJPMJMS2024 Japan
Japan Agency for Medical Research and Development (AMED)JP21gm1610003 Japan
Japan Society for the Promotion of Science (JSPS)22K06809 Japan
CitationJournal: bioRxiv / Year: 2024
Title: Structural transitions in kinesin minus-end directed microtubule motility.
Authors: Satoki Shibata / Matthew Y Wang / Tsuyoshi Imasaki / Hideki Shigematsu / Yuanyuan Wei / Chacko Jobichen / Hajime Hagio / J Sivaraman / Sharyn A Endow / Ryo Nitta /
Abstract: Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for ...Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central β-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with β-strand-to-loop transitions, implying that β-strand refolding induces Pi release and the recovery stroke. The strained β-sheet during the power stroke and strand-to-loop transitions identify the β-sheet as the long-sought motor spring.
History
DepositionApr 3, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 8, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
C: Protein claret segregational
D: Protein claret segregational
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,2418
Polymers192,8234
Non-polymers1,4184
Water00
1
A: Tubulin alpha-1B chain
B: Tubulin beta chain
C: Protein claret segregational
D: Protein claret segregational
hetero molecules
x 14


Theoretical massNumber of molelcules
Total (without water)2,719,372112
Polymers2,699,52256
Non-polymers19,85056
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation13

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Components

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Protein , 3 types, 4 molecules ABCD

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


Mass: 50204.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
References: UniProt: Q2XVP4, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Protein Tubulin beta chain / Tubulin beta-5 chain


Mass: 49717.629 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q767L7
#3: Protein Protein claret segregational / Kinesin-14


Mass: 46450.453 Da / Num. of mol.: 2 / Mutation: E292M, Y485K, N697S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: ncd, CA(ND), CG7831 / Production host: Escherichia coli (E. coli)
References: UniProt: P20480, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Kinesin-microtubule complexCOMPLEX#1-#30MULTIPLE SOURCES
2TubulinCOMPLEX#1-#21NATURAL
3NCDCOMPLEX#31RECOMBINANT
Molecular weightValue: 200 kDa/nm / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa (pig)9823
23Drosophila melanogaster (fruit fly)7227
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
Details: 100 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA, and 1 mM GTP
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMPIPESPIPES1
21 mMMgCl2MgCl21
31 mMEGTAEGTA1
41 mMGTPGTP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 10 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 310 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4RELION3.1.4CTF correction
8PHENIXmodel refinement
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: -25.7415 ° / Axial rise/subunit: 8.75508 Å / Axial symmetry: C13
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45321 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612915
ELECTRON MICROSCOPYf_angle_d0.65617500
ELECTRON MICROSCOPYf_dihedral_angle_d14.2984801
ELECTRON MICROSCOPYf_chiral_restr0.0441952
ELECTRON MICROSCOPYf_plane_restr0.0052274

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