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- PDB-6vpp: Cryo-EM structure of microtubule-bound KLP61F motor with tail dom... -

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Basic information

Entry
Database: PDB / ID: 6vpp
TitleCryo-EM structure of microtubule-bound KLP61F motor with tail domain in the nucleotide-free state
Components
  • Kinesin-like protein Klp61F
  • Tubulin alpha-1A chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN / Kinesin-5 / KLP61F / microtubules / mitotic spindles / nucleotide-free state / cell cycle
Function / homology
Function and homology information


aster / plus-end directed microtubule sliding / fusome organization / fusome / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centrosome separation / spindle elongation / mitotic spindle microtubule / positive regulation of Golgi to plasma membrane protein transport ...aster / plus-end directed microtubule sliding / fusome organization / fusome / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centrosome separation / spindle elongation / mitotic spindle microtubule / positive regulation of Golgi to plasma membrane protein transport / microtubule bundle formation / plus-end-directed microtubule motor activity / mitotic centrosome separation / microtubule associated complex / kinesin complex / microtubule-based movement / mitotic spindle pole / Golgi organization / cytoskeletal motor activity / protein secretion / mitotic spindle assembly / mitotic spindle organization / spindle microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / mitotic spindle / spindle / microtubule cytoskeleton organization / mitotic cell cycle / microtubule binding / microtubule / hydrolase activity / cell division / GTPase activity / GTP binding / Golgi apparatus / endoplasmic reticulum / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / 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-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / 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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein Klp61F
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBodrug, T. / Wilson-Kubalek, E.M. / Nithianantham, S. / Debs, G. / Sindelar, C.V. / Milligan, R. / Al-Bassam, J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM110283 United States
National Science Foundation (NSF, United States)1615991 United States
CitationJournal: Elife / Year: 2020
Title: The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.
Authors: Tatyana Bodrug / Elizabeth M Wilson-Kubalek / Stanley Nithianantham / Alex F Thompson / April Alfieri / Ignas Gaska / Jennifer Major / Garrett Debs / Sayaka Inagaki / Pedro Gutierrez / ...Authors: Tatyana Bodrug / Elizabeth M Wilson-Kubalek / Stanley Nithianantham / Alex F Thompson / April Alfieri / Ignas Gaska / Jennifer Major / Garrett Debs / Sayaka Inagaki / Pedro Gutierrez / Larisa Gheber / Richard J McKenney / Charles Vaughn Sindelar / Ronald Milligan / Jason Stumpff / Steven S Rosenfeld / Scott T Forth / Jawdat Al-Bassam /
Abstract: Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a ...Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
History
DepositionFeb 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 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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Structure visualization

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Assembly

Deposited unit
A: Tubulin alpha-1A chain
B: Tubulin beta chain
C: Kinesin-like protein Klp61F
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,6476
Polymers142,6563
Non-polymers9913
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7940 Å2
ΔGint-45 kcal/mol
Surface area45750 Å2

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Tubulin alpha-1A chain / / Alpha-tubulin 1 / Tubulin alpha-1 chain


Mass: 50121.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02550
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein Kinesin-like protein Klp61F / Bipolar kinesin KRP-130


Mass: 42627.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Klp61F, KLP2, CG9191 / Production host: Escherichia coli (E. coli) / References: UniProt: P46863

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Non-polymers , 3 types, 3 molecules

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

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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: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Microtubule-bound KLP61F motor with tail domain in the nucleotide-free stateCOMPLEX#1-#30MULTIPLE SOURCES
2microtubuleCOMPLEX#1-#21NATURAL
3KLP61F motorCOMPLEX#31RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / 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
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K

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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 FIELDBright-field microscopy
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10FREALIGNinitial Euler assignment
12RELIONclassification
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -34.61 ° / Axial rise/subunit: 17.4 Å / Axial symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14570 / Symmetry type: HELICAL
Atomic model buildingB value: 208 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

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