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- EMDB-3778: Molecular basis of human kinesin-8 function and inhibition -

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Entry
Database: EMDB / ID: EMD-3778
TitleMolecular basis of human kinesin-8 function and inhibition
Map dataAsymmetric unit extracted from the full MT-bound Kif18A motor domain no nucleotide reconstruction
Sample
  • Complex: 13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide
    • Complex: Tubulin, alpha
      • Protein or peptide: Tubulin alpha chain
    • Complex: Tublin, beta
      • Protein or peptide: Tubulin beta chain
    • Complex: Kinesin-8 motor domain neck-linker
      • Protein or peptide: Kinesin-like protein KIF18A
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL
KeywordsCytoskeleton / Motor protein / Kinesin / TRANSPORT PROTEIN
Function / homology
Function and homology information


tubulin-dependent ATPase activity / mitotic spindle astral microtubule / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase ...tubulin-dependent ATPase activity / mitotic spindle astral microtubule / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / COPI-dependent Golgi-to-ER retrograde traffic / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / MHC class II antigen presentation / mitotic spindle midzone / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Aggrephagy / kinetochore microtubule / The role of GTSE1 in G2/M progression after G2 checkpoint / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / male meiotic nuclear division / Recruitment of NuMA to mitotic centrosomes / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / microtubule plus-end binding / Kinesins / plus-end-directed microtubule motor activity / microtubule depolymerization / COPI-dependent Golgi-to-ER retrograde traffic / kinesin complex / mitotic metaphase chromosome alignment / microtubule-based movement / seminiferous tubule development / mitotic sister chromatid segregation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / regulation of microtubule cytoskeleton organization / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / ruffle / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / cellular response to estradiol stimulus / RHO GTPases Activate Formins / caveola / structural constituent of cytoskeleton / kinetochore / microtubule cytoskeleton organization / Separation of Sister Chromatids / microtubule cytoskeleton / protein transport / mitotic cell cycle / actin binding / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / centrosome / GTP binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / 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
Tubulin alpha chain / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin-like protein KIF18A
Similarity search - Component
Biological speciesBos taurus (cattle) / Sus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsLocke J / Joseph AP
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Cancer Research UKC33336/A13177 United Kingdom
Worldwide Cancer Research16-0037 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Structural basis of human kinesin-8 function and inhibition.
Authors: Julia Locke / Agnel Praveen Joseph / Alejandro Peña / Martin M Möckel / Thomas U Mayer / Maya Topf / Carolyn A Moores /
Abstract: Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their ...Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end-directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1-bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1-binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.
History
DepositionJun 23, 2017-
Header (metadata) releaseJul 26, 2017-
Map releaseOct 25, 2017-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5oam
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3778.png

Downloads & links

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Map

FileDownload / File: emd_3778.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric unit extracted from the full MT-bound Kif18A motor domain no nucleotide reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 75 pix.
= 104.25 Å		1.39 Å/pix.
x 57 pix.
= 79.23 Å		1.39 Å/pix.
x 77 pix.
= 107.03 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.39 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum0.0 - 0.17798981
Average (Standard dev.)0.020312501 (±0.033570096)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin20489143
Dimensions577775
Spacing775775
CellA: 107.03 Å / B: 79.229996 Å / C: 104.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z775775
origin x/y/z0.0000.0000.000
length x/y/z107.03079.230104.250
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS89204143
NC/NR/NS775775
D min/max/mean0.0000.1780.020

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Supplemental data

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Sample components

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Entire : 13-Protofilament microtuble-bound human Kinesin-8 motor domain ne...

EntireName: 13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide
Components
  • Complex: 13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide
    • Complex: Tubulin, alpha
      • Protein or peptide: Tubulin alpha chain
    • Complex: Tublin, beta
      • Protein or peptide: Tubulin beta chain
    • Complex: Kinesin-8 motor domain neck-linker
      • Protein or peptide: Kinesin-like protein KIF18A
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL

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Supramolecule #1: 13-Protofilament microtuble-bound human Kinesin-8 motor domain ne...

SupramoleculeName: 13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Tubulin, alpha

SupramoleculeName: Tubulin, alpha / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)

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Supramolecule #3: Tublin, beta

SupramoleculeName: Tublin, beta / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #4: Kinesin-8 motor domain neck-linker

SupramoleculeName: Kinesin-8 motor domain neck-linker / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Kinesin-like protein KIF18A

MacromoleculeName: Kinesin-like protein KIF18A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.213938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMSVTEED LCHHMKVVVR VRPENTKEKA AGFHKVVHVV DKHILVFDPK QEEVSFFHGK KTTNQNVIKK QNKDLKFVFD AVFDETSTQ SEVFEHTTKP ILRSFLNGYN CTVLAYGATG AGKTHTMLGS ADEPGVMYLT MLHLYKCMDE IKEEKICSTA V SYLEVYNE ...String:
GSHMSVTEED LCHHMKVVVR VRPENTKEKA AGFHKVVHVV DKHILVFDPK QEEVSFFHGK KTTNQNVIKK QNKDLKFVFD AVFDETSTQ SEVFEHTTKP ILRSFLNGYN CTVLAYGATG AGKTHTMLGS ADEPGVMYLT MLHLYKCMDE IKEEKICSTA V SYLEVYNE QIRDLLVNSG PLAVREDTQK GVVVHGLTLH QPKSSEEILH LLDNGNKNRT QHPTDMNATS SRSHAVFQIY LR QQDKTAS INQNVRIAKM SLIDLAGSER ASTSGAKGTR FVEGTNINRS LLALGNVINA LADSKRKNQH IPYRNSKLTR LLK DSLGGN CQTIMIAAVS PSSVFYDDTY NTLKYANRAK DIKSSLKSNV LNVNNHITQY V

UniProtKB: Kinesin-like protein KIF18A

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Macromolecule #2: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 50.107238 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 NLVPYPRGHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha chain

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Macromolecule #3: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 3 / 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 #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Macromolecule #7: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

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

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Macromolecule #8: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 8 / Number of copies: 1 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridModel: C-flat-2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF Quantum
DetailsFEI Polara
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Average exposure time: 2.0 sec. / Average electron dose: 5.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 81.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 70395
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2p4n

Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5oam:
Molecular basis of human kinesin-8 function and inhibition

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