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

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Entry
Database: EMDB / ID: 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
Sample13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide:
Tubulin, alpha / Tublin, beta / Kinesin-8 motor domain neck-linker / Kinesin-like protein KIF18A / Tubulin alpha chain / Tubulin beta chain / (ligand) x 5
Function / homologyTubulin, conserved site / Kinesin-like protein / Recruitment of NuMA to mitotic centrosomes / Tubulin / Kinesin motor domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site ...Tubulin, conserved site / Kinesin-like protein / Recruitment of NuMA to mitotic centrosomes / Tubulin / Kinesin motor domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Recruitment of mitotic centrosome proteins and complexes / Tubulin/FtsZ, 2-layer sandwich domain / Kinesin motor domain, conserved site / Tubulin, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Tubulin/FtsZ, GTPase domain superfamily / Recycling pathway of L1 / Kinesin motor domain superfamily / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ family, GTPase domain / Kinesin motor domain / Tubulin C-terminal domain / Tubulin subunits alpha, beta, and gamma signature. / Tubulin-beta mRNA autoregulation signal. / Kinesin motor domain signature. / Kinesin motor domain profile. / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / MHC class II antigen presentation / Separation of Sister Chromatids / Resolution of Sister Chromatid Cohesion / Regulation of PLK1 Activity at G2/M Transition / Loss of Nlp from mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Anchoring of the basal body to the plasma membrane / Kinesins / RHO GTPases Activate Formins / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / MHC class II antigen presentation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Kinesins / Carboxyterminal post-translational modifications of tubulin / AURKA Activation by TPX2 / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / COPI-dependent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / Intraflagellar transport / COPI-mediated anterograde transport / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / tubulin-dependent ATPase activity / mitotic spindle midzone / kinetochore microtubule / male meiotic nuclear division / C-terminal protein amino acid modification / microtubule plus-end binding / microtubule depolymerization / seminiferous tubule development / ATP-dependent microtubule motor activity, plus-end-directed / mitotic metaphase plate congression / regulation of microtubule cytoskeleton organization / kinesin complex / retrograde vesicle-mediated transport, Golgi to ER / cytoplasmic microtubule / cellular response to interleukin-4 / microtubule-based process / recycling endosome / sister chromatid cohesion / microtubule cytoskeleton organization / antigen processing and presentation of exogenous peptide antigen via MHC class II / kinetochore / cytoplasmic ribonucleoprotein granule / structural constituent of cytoskeleton / microtubule-based movement / ruffle / microtubule cytoskeleton / cellular response to estradiol stimulus / microtubule organizing center / caveola / double-stranded RNA binding / protein transport / actin binding / microtubule / microtubule binding / GTPase activity / myelin sheath / ubiquitin protein ligase binding / GTP binding / ATP binding / nucleus / cytosol / cytoplasm / Tubulin alpha chain / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin-like protein KIF18A
Function and homology information
SourceBos taurus (cattle) / Sus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / 5.5 Å resolution
AuthorsLocke J / Joseph AP
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.
Validation ReportPDB-ID: 5oam

SummaryFull reportAbout validation report
DateDeposition: Jun 23, 2017 / Header (metadata) release: Jul 26, 2017 / Map release: Oct 25, 2017 / Last update: Nov 15, 2017

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Structure visualization

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

Downloads & links

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Map

Fileemd_3778.map.gz (map file in CCP4 format, 1317 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
75 pix
1.39 Å/pix.
= 104.25 Å
57 pix
1.39 Å/pix.
= 79.23 Å
77 pix
1.39 Å/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
Contour Level:0.05 (by author), 0.05 (movie #1):
Minimum - Maximum0 - 0.17798981
Average (Standard dev.)0.020312501 (0.033570096)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions577775
Origin20489143
Limit260165217
Spacing775775
CellA: 107.03 Å / B: 79.229996 Å / C: 104.25 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
Number of components: 12

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Component #1: protein, 13-Protofilament microtuble-bound human Kinesin-8 motor ...

ProteinName: 13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker without nucleotide
Recombinant expression: No

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Component #2: protein, Tubulin, alpha

ProteinName: Tubulin, alpha / Recombinant expression: No
SourceSpecies: Bos taurus (cattle)

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Component #3: protein, Tublin, beta

ProteinName: Tublin, beta / Recombinant expression: No
SourceSpecies: Sus scrofa (pig)

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Component #4: protein, Kinesin-8 motor domain neck-linker

ProteinName: Kinesin-8 motor domain neck-linker / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, Kinesin-like protein KIF18A

ProteinName: Kinesin-like protein KIF18A / Recombinant expression: No
MassTheoretical: 42.213938 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, Tubulin alpha chain

ProteinName: Tubulin alpha chain / Recombinant expression: No
MassTheoretical: 50.107238 kDa
SourceSpecies: Bos taurus (cattle)

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Component #7: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Recombinant expression: No
MassTheoretical: 49.90777 kDa
SourceSpecies: Sus scrofa (pig)

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Component #8: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #9: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #10: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Component #11: ligand, GUANOSINE-5'-DIPHOSPHATE

LigandName: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.443201 kDa

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Component #12: ligand, TAXOL

LigandName: TAXOLPaclitaxel / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.853906 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 81 Å
Sample solutionpH: 7.4
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Details: FEI Polara
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 5 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.3 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - nm / Energy filter: GIF Quantum
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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