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- PDB-7tr2: Apo CaKip3[2-436]-L2-mutant(HsKHC) in complex with a microtubule -

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Basic information

Entry
Database: PDB / ID: 7tr2
TitleApo CaKip3[2-436]-L2-mutant(HsKHC) in complex with a microtubule
Components
  • Kinesin-like protein,Kinesin-1 heavy chain
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / Kip3 / kinesin / motility / microtubule / tubulin
Function / homology
Function and homology information


regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet ...regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet / lysosome localization / positive regulation of potassium ion transport / plus-end-directed microtubule motor activity / vesicle transport along microtubule / RHO GTPases activate KTN1 / Kinesins / 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 / kinesin complex / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / microtubule motor activity / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / centrosome localization / mitochondrion transport along microtubule / COPI-mediated anterograde transport / COPI-dependent Golgi-to-ER retrograde traffic / microtubule-based movement / stress granule disassembly / natural killer cell mediated cytotoxicity / Insulin processing / synaptic vesicle transport / postsynaptic cytosol / microtubule-based process / phagocytic vesicle / axon cytoplasm / MHC class II antigen presentation / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / regulation of membrane potential / positive regulation of protein localization to plasma membrane / cellular response to type II interferon / structural constituent of cytoskeleton / microtubule cytoskeleton organization / centriolar satellite / Signaling by ALK fusions and activated point mutants / mitotic cell cycle / microtubule cytoskeleton / nuclear membrane / microtubule binding / vesicle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cadherin binding / GTPase activity / GTP binding / protein-containing complex binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
Kinesin-like protein / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 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-like protein / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin beta chain / Kinesin-like protein / Kinesin-1 heavy chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesCandida albicans (yeast)
Homo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 Å
AuthorsBenoit, M.P.M.H. / Asenjo, A.B. / Hunter, B. / Allingham, J.S. / Sosa, H.
Funding support United States, Canada, 3items
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/356025-2013 Canada
Canadian Institutes of Health Research (CIHR)MOP-97832 Canada
CitationJournal: Nat Commun / Year: 2022
Title: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape.
Authors: Byron Hunter / Matthieu P M H Benoit / Ana B Asenjo / Caitlin Doubleday / Daria Trofimova / Corey Frazer / Irsa Shoukat / Hernando Sosa / John S Allingham /
Abstract: Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains ...Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic. We also determined a crystal structure of microtubule-unbound CaKip3-ADP (2.0 Å) and analyzed the biochemical activity of CaKip3 and kinesin-1 mutants. These data reveal that the microtubule depolymerization activity of kinesin-8 originates from conformational changes of its motor core that are amplified by dynamic contacts between its extended loop-2 and tubulin. On curved microtubule ends, loop-1 inserts into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin and assist depolymerization. On straight tubulin protofilaments in the microtubule lattice, loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. We propose that these tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.
History
DepositionJan 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
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
Revision 1.2Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)148,3947
Polymers146,5493
Non-polymers1,8454
Water00
1


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

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Components

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Protein , 3 types, 3 molecules KAB

#1: Protein Kinesin-like protein,Kinesin-1 heavy chain / Conventional kinesin heavy chain / Ubiquitous kinesin heavy chain / UKHC


Mass: 46344.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CaKip3[2-436] with L2 swapped with the one of HsKHC,CaKip3[2-436] with L2 swapped with the one of HsKHC,CaKip3[2-436] with L2 swapped with the one of HsKHC
Source: (gene. exp.) Candida albicans (yeast), (gene. exp.) Homo sapiens (human)
Gene: KIF5B, KNS, KNS1 / Production host: Escherichia coli (E. coli) / References: UniProt: C4YNU9, UniProt: P33176
#2: 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
#3: 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

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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 / 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*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
IDNameTypeDetailsEntity IDParent-IDSource
1Apo CaKip3[2-436]-L2-mutant(HsKHC) in complex with a microtubuleCOMPLEXCaKip3[2-436] has it's L2 swapped with the one of HsKHC#1-#30MULTIPLE SOURCES
2CaKip3[2-436] with L2 swapped with the one of HsKHC, bound to AMP-PNPCOMPLEX#11RECOMBINANT
315R microtubuleORGANELLE OR CELLULAR COMPONENT#2-#31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
12Candida albicans (yeast)5476
23Sus scrofa (pig)9823Brain
32Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameBuffer-ID
180 mMK-PIPES1
220 uMPaclitaxel1
31 mMMagnesium chloride1
41 mMEGTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R2/2
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 defocus max: 1500 nm / Nominal defocus min: 700 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.6 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
11Cootmodel fitting
14SPIDER22.1initial Euler assignment
15RELION3.1final Euler assignment
17RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 168.088 ° / Axial rise/subunit: 5.55 Å / Axial symmetry: C1
Particle selectionDetails: manual picking of filaments
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98446
Details: 2 half datasets containing one distinct half of each filament were refined independently. Number of asymmetric units used is reported in "number of segments used", due to the local processing strategy employed.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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