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- EMDB-3803: Molecular basis of human kinesin-8 function and inhibition -

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Entry
Database: EMDB / ID: 3803
TitleMolecular basis of human kinesin-8 function and inhibition
Map dataAsymmetric unit extracted from the full MT-bound Kif18A motor domain, BTB-1-bound reconstruction
Sample13-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker with its specific inhibitor BTB-1:
Kinesin-8 motor domain neck-linker / (Tubulin alpha ...) x 2 / (Tubulin beta ...) x 2 / Kinesin-like protein KIF18A / (ligand) x 6
Function / homologyBeta tubulin, autoregulation binding site / Tubulin, conserved site / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Loss of Nlp from mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Regulation of PLK1 Activity at G2/M Transition / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Tubulin / Kinesin motor domain ...Beta tubulin, autoregulation binding site / Tubulin, conserved site / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Loss of Nlp from mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Regulation of PLK1 Activity at G2/M Transition / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Tubulin / Kinesin motor domain / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / MHC class II antigen presentation / Tubulin/FtsZ, 2-layer sandwich domain / Recycling pathway of L1 / Kinesin motor domain, conserved site / Tubulin, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Kinesin-like protein / Tubulin/FtsZ, GTPase domain superfamily / 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. / Recruitment of NuMA to mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Hedgehog 'off' state / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / COPI-independent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / The role of GTSE1 in G2/M progression after G2 checkpoint / AURKA Activation by TPX2 / Carboxyterminal post-translational modifications of tubulin / Kinesins / MHC class II antigen presentation / Cilium Assembly / Separation of Sister Chromatids / Resolution of Sister Chromatid Cohesion / RHO GTPases Activate Formins / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / RHO GTPases activate IQGAPs / Anchoring of the basal body to the plasma membrane / Intraflagellar transport / Kinesin motor domain profile. / Hedgehog 'on' state / tubulin-dependent ATPase activity / mitotic spindle astral microtubule / kinetochore microtubule / mitotic spindle midzone / male meiotic nuclear division / microtubule depolymerization / microtubule plus-end binding / ATP-dependent microtubule motor activity, plus-end-directed / mitotic metaphase plate congression / regulation of synapse organization / kinesin complex / microtubule motor activity / seminiferous tubule development / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / regulation of microtubule cytoskeleton organization / mitotic sister chromatid segregation / cytoplasmic microtubule / cellular response to interleukin-4 / microtubule-based movement / recycling endosome / microtubule-based process / antigen processing and presentation of exogenous peptide antigen via MHC class II / kinetochore / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / ruffle / microtubule cytoskeleton organization / neuron migration / cellular response to estradiol stimulus / microtubule cytoskeleton / microtubule organizing center / caveola / neuromuscular junction / protein transport / double-stranded RNA binding / actin binding / mitotic cell cycle / microtubule / microtubule binding / ATPase activity / GTPase activity / myelin sheath / GTP binding / ubiquitin protein ligase binding
Function and homology information
SourceHomo sapiens (human) / Bos taurus (cattle) / sus scrofa (pig) / Bovine (cattle) / Pig (pig)
Methodhelical reconstruction / cryo EM / 4.8 Å resolution
AuthorsLocke J / Joseph AP / Topf M / Moores CA
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: 5ogc

SummaryFull reportAbout validation report
DateDeposition: Jul 12, 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
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  • Surface view with fitted model
  • Atomic models: : PDB-5ogc
  • 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_3803.map.gz (map file in CCP4 format, 1410 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
75 pix
1.39 Å/pix.
= 104.25 Å
58 pix
1.39 Å/pix.
= 80.62 Å
81 pix
1.39 Å/pix.
= 112.59 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 - 0.20442685
Average (Standard dev.)0.019545615 (0.034245346)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions588175
Origin203.087.0145.0
Limit260.0167.0219.0
Spacing815875
CellA: 112.59 Å / B: 80.62 Å / C: 104.25 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z815875
origin x/y/z0.0000.0000.000
length x/y/z112.59080.620104.250
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS87203145
NC/NR/NS815875
D min/max/mean0.0000.2040.020

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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 with its specific inhibitor BTB-1
Number of components: 13

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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 with its specific inhibitor BTB-1
Recombinant expression: No

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Component #2: 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 #3: protein, Tubulin alpha chain

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

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Component #4: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Recombinant expression: No
SourceSpecies: sus scrofa (pig)

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Component #5: protein, Kinesin-like protein KIF18A

ProteinName: Kinesin-like protein KIF18A / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 42.213938 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: protein, Tubulin alpha chain

ProteinName: Tubulin alpha chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 50.107238 kDa
SourceSpecies: Bovine (cattle)

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Component #7: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.90777 kDa
SourceSpecies: Pig (pig)

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Component #8: ligand, 4-chloranyl-2-nitro-1-(phenylsulfonyl)benzene

LigandName: 4-chloranyl-2-nitro-1-(phenylsulfonyl)benzene / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.297714 kDa

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Component #9: ligand, ZINC ION

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

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Component #10: ligand, MAGNESIUM ION

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

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Component #11: 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 #12: ligand, GUANOSINE-5'-DIPHOSPHATE

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

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Component #13: 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 / Delta phi: 0 deg.
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: 500.0 - nm / Energy filter: GIF Quantum
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Output model

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