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- EMDB-21314: Cryo-EM structure of microtubule-bound KLP61F motor domain in the... -

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Basic information

Entry
Database: EMDB / ID: EMD-21314
TitleCryo-EM structure of microtubule-bound KLP61F motor domain in the AMPPNP state
Map datamicrotubule-bound KLP61F motor domain in the AMPPNP state
Sample
  • Complex: Microtubule-bound KLP61F motor in the AMPPNP state
    • Complex: microtubule
      • Protein or peptide: Tubulin alpha-1A chain
      • Protein or peptide: Tubulin beta chain
    • Complex: KLP61F motor
      • Protein or peptide: Kinesin-like protein Klp61F
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsKinesin-5 / microtubules / mitotic spindles / AMPPNP state / cell cycle / MOTOR PROTEIN
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
Tubulin alpha-1A chain / Tubulin beta chain / Kinesin-like protein Klp61F
Similarity search - Component
Biological speciesSus scrofa (pig) / Drosophila melanogaster (fruit fly)
Methodhelical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBodrug T / Wilson-Kubalek EM
Funding support United States, 2 items
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, 2020-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
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  • Surface view colored by height
  • Surface level: 0.025
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  • Surface view with fitted model
  • Atomic models: PDB-6vpo
  • Surface level: 0.025
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vpo
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21314.png

Downloads & links

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Map

FileDownload / File: emd_21314.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmicrotubule-bound KLP61F motor domain in the AMPPNP state
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.056669515 - 0.109821804
Average (Standard dev.)0.0019629127 (±0.015592641)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin250142126
Dimensions1346479
Spacing6413479
CellA: 83.84 Å / B: 175.54 Å / C: 103.49 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z6413479
origin x/y/z0.0000.0000.000
length x/y/z83.840175.540103.490
α/β/γ90.00090.00090.000
start NX/NY/NZ-532-20
NX/NY/NZ1019393
MAP C/R/S123
start NC/NR/NS142250126
NC/NR/NS6413479
D min/max/mean-0.0570.1100.002

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Supplemental data

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Sample components

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Entire : Microtubule-bound KLP61F motor in the AMPPNP state

EntireName: Microtubule-bound KLP61F motor in the AMPPNP state
Components
  • Complex: Microtubule-bound KLP61F motor in the AMPPNP state
    • Complex: microtubule
      • Protein or peptide: Tubulin alpha-1A chain
      • Protein or peptide: Tubulin beta chain
    • Complex: KLP61F motor
      • Protein or peptide: Kinesin-like protein Klp61F
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Microtubule-bound KLP61F motor in the AMPPNP state

SupramoleculeName: Microtubule-bound KLP61F motor in the AMPPNP state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: microtubule

SupramoleculeName: microtubule / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: KLP61F motor

SupramoleculeName: KLP61F motor / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: Tubulin alpha-1A chain

MacromoleculeName: Tubulin alpha-1A chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 50.121266 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRAHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA

UniProtKB: Tubulin beta chain

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Macromolecule #3: Kinesin-like protein Klp61F

MacromoleculeName: Kinesin-like protein Klp61F / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 42.627336 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDISGGNTSR QPQKKSNQNI QVYVRVRPLN SRERCIRSAE VVDVVGPREV VTRHTLDSKL TKKFTFDRSF GPESKQCDVY SVVVSPLIE EVLNGYNCTV FAYGQTGTGK THTMVGNETA ELKSSWEDDS DIGIIPRALS HLFDELRMME VEYTMRISYL E LYNEELCD ...String:
MDISGGNTSR QPQKKSNQNI QVYVRVRPLN SRERCIRSAE VVDVVGPREV VTRHTLDSKL TKKFTFDRSF GPESKQCDVY SVVVSPLIE EVLNGYNCTV FAYGQTGTGK THTMVGNETA ELKSSWEDDS DIGIIPRALS HLFDELRMME VEYTMRISYL E LYNEELCD LLSTDDTTKI RIFDDSTKKG SVIIQGLEEI PVHSKDDVYK LLEKGKERRK TATTLMNAQS SRSHTVFSIV VH IRENGIE GEDMLKIGKL NLVDLAGSEN VSKAGNEKGI RVRETVNINQ SLLTLGRVIT ALVDRAPHVP YRESKLTRLL QES LGGRTK TSIIATISPG HKDIEETLST LEYAHRAKNI QNKPEVNQKL TKKLEHHHHH H

UniProtKB: Kinesin-like protein Klp61F

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Macromolecule #7: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 6.8
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 38.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 73451 / Software - Name: FREALIGN
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 17.4 Å
Applied symmetry - Helical parameters - Δ&Phi: -34.61 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39220

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 216 / Target criteria: Correlation coefficient
Output model

PDB-6vpo:
Cryo-EM structure of microtubule-bound KLP61F motor domain in the AMPPNP state

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