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- PDB-6wwr: Kif14[391-743] - ADP-AlFx open state class in complex with a micr... -

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Basic information

Entry
Database: PDB / ID: 6wwr
TitleKif14[391-743] - ADP-AlFx open state class in complex with a microtubule
Components
  • Kinesin-like protein KIF14
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / KIF14 / kinesin / motility / microtubule / tubulin
Function / homology
Function and homology information


cerebellar granular layer structural organization / regulation of cell maturation / cerebellar Purkinje cell layer structural organization / RHO GTPases activate CIT / negative regulation of integrin activation / RND2 GTPase cycle / RND1 GTPase cycle / cell proliferation in forebrain / regulation of Rap protein signal transduction / cerebellar cortex development ...cerebellar granular layer structural organization / regulation of cell maturation / cerebellar Purkinje cell layer structural organization / RHO GTPases activate CIT / negative regulation of integrin activation / RND2 GTPase cycle / RND1 GTPase cycle / cell proliferation in forebrain / regulation of Rap protein signal transduction / cerebellar cortex development / olfactory bulb development / 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 / Flemming body / plus-end-directed microtubule motor activity / 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 / microtubule motor activity / regulation of myelination / kinesin complex / mitotic metaphase chromosome alignment / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / activation of protein kinase activity / microtubule-based movement / positive regulation of cytokinesis / regulation of G1/S transition of mitotic cell cycle / spindle midzone / regulation of cell adhesion / regulation of neuron apoptotic process / regulation of G2/M transition of mitotic cell cycle / regulation of cell migration / tubulin binding / substrate adhesion-dependent cell spreading / hippocampus development / regulation of cell growth / PDZ domain binding / establishment of protein localization / structural constituent of cytoskeleton / cerebral cortex development / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / midbody / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / microtubule / cell division / GTPase activity / positive regulation of cell population proliferation / negative regulation of apoptotic process / GTP binding / protein kinase binding / ATP hydrolysis activity / ATP binding / membrane / nucleus / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Kinesin-associated / Kinesin-associated / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. ...Kinesin-associated / Kinesin-associated / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / SMAD/FHA domain superfamily / 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 / TAXOL / Tubulin beta chain / Kinesin-like protein KIF14 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBenoit, M.P.M.H. / Asenjo, A.B. / Paydar, M. / Dhakal, S. / Kwok, B. / Sosa, H.
Funding support United States, Canada, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM113164 United States
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN/04103-2015 Canada
Canadian Cancer Society Research Institute703405 Canada
Fonds de Recherche du Quebec-Sante (FRSQ)Chercheure-Boursiere Junior 1, Junior 2 Awards Canada
Canadian Institutes of Health Research (CIHR)New Investigator Award Canada
Simons FoundationSF349247 United States
NYSTAR United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14.
Authors: Matthieu P M H Benoit / Ana B Asenjo / Mohammadjavad Paydar / Sabin Dhakal / Benjamin H Kwok / Hernando Sosa /
Abstract: KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding ...KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7-3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.
History
DepositionMay 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 7, 2021Group: Author supporting evidence / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_audit_support / struct_sheet
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _struct_sheet.number_strands
Revision 1.2May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta-2B chain
K: Kinesin-like protein KIF14
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,2288
Polymers139,9563
Non-polymers2,2725
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABK

#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) / Organ: Brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta-2B chain


Mass: 49999.887 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Brain / References: UniProt: A0A287AGU7
#3: Protein Kinesin-like protein KIF14


Mass: 39751.730 Da / Num. of mol.: 1 / Fragment: UNP residues 391-743
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kif14 / Production host: Escherichia coli (E. coli) / References: UniProt: L0N7N1

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Non-polymers , 5 types, 5 molecules

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


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


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

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Details

Has ligand of interestN

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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
1Kif14[391-743] - ADP-AlFx in complex with a microtubuleCOMPLEX#1-#30MULTIPLE SOURCES
2KIF14[391-743] - ADP-AlFxCOMPLEX#31RECOMBINANT
3MicrotubuleORGANELLE OR CELLULAR COMPONENT#1-#21NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Sus scrofa (pig)9823
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameBuffer-ID
180 mMK-PIPES1
220 uMPaclitaxel1
35 mMMagnesium chloride1
41 mMEGTA1
54 mMADP1
62 mMAlCl31
710 mMKF1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 60606 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
2Leginonbetaimage acquisition
4RELION3.1CTF correction
7RosettaEMmodel fitting
8PHENIXmodel fitting
9MODELLERmodel fittingUsed through UCSF chimera
10UCSF Chimeramodel fitting
11Coot0.8.9model fitting
14SPIDER22.1initial Euler assignment
15RELION3.1final Euler assignment
17RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 168.07 ° / Axial rise/subunit: 5.44 Å / Axial symmetry: C1
Particle selectionDetails: manual picking of filaments
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 348209 / Algorithm: FOURIER SPACE
Details: 2 half datasets containing one distinct half of each filament were refined independently.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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