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- PDB-7tr3: CaKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilize... -

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Basic information

Entry
Database: PDB / ID: 7tr3
TitleCaKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
Components
  • Kinesin-like protein
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / Kip3 / kinesin / tubulin / dolastatin-10
Function / homology
Function and homology information


tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / mitotic spindle astral microtubule / meiotic sister chromatid segregation / nuclear microtubule / mitotic spindle midzone / nuclear migration along microtubule / microtubule plus-end / microtubule nucleation ...tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / mitotic spindle astral microtubule / meiotic sister chromatid segregation / nuclear microtubule / mitotic spindle midzone / nuclear migration along microtubule / microtubule plus-end / microtubule nucleation / mitotic spindle disassembly / 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 / plus-end-directed microtubule motor activity / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / kinesin complex / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / microtubule depolymerization / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / COPI-mediated anterograde transport / microtubule-based movement / negative regulation of microtubule polymerization / establishment of mitotic spindle orientation / mitotic sister chromatid segregation / mitotic spindle assembly / microtubule-based process / mitotic spindle organization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Kinesin-like protein / 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-like protein / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / dolastatin-10 / Kinesin-like protein / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesCandida albicans (yeast)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 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
A: Tubulin alpha-1B chain
B: Tubulin beta-2B chain
K: Kinesin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,3568
Polymers155,0743
Non-polymers2,2825
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 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) / References: UniProt: A0A287AGU7
#3: Protein Kinesin-like protein


Mass: 54869.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candida albicans (yeast) / Gene: KIP3, orf19.7353, CAALFM_C305720CA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1D8PKA4

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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-SR6 / dolastatin-10 / N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide


Mass: 785.091 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H68N6O6S / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ringCOMPLEX#1-#30MULTIPLE SOURCES
2caKip3[2-482] - AMP-PNPCOMPLEX#31RECOMBINANT
3dolastatin-10 stabilized tubulin ringORGANELLE OR CELLULAR COMPONENT#1-#21NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
22Candida albicans (yeast)5476
33Sus scrofa (pig)9823Brain
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameBuffer-ID
180 mMK-PIPES1
220 uMDolastatin-101
35 mMMagnesium chloride1
41 mMEGTA1
54 mMAMP-PNP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid 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: 2670 nm / Nominal defocus min: 810 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64.01 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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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
14RELION3.1initial Euler assignment
15RELION3.1final Euler assignment
17RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C14 (14 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 530936
Details: The "Number of particles used" reported here is the number of asymmetric units used.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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