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- EMDB-10422: Human kinesin-5 motor domain in the AMPPNP state bound to microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-10422
TitleHuman kinesin-5 motor domain in the AMPPNP state bound to microtubules
Map data
Sample
  • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
    • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
      • Protein or peptide: Tubulin beta chain
      • Protein or peptide: Tubulin alpha-1B chain
    • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
      • Protein or peptide: Kinesin-like protein KIF11
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Function / homology
Function and homology information


spindle elongation / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins ...spindle elongation / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Kinesins / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / plus-end-directed microtubule motor activity / Recruitment of NuMA to mitotic centrosomes / regulation of mitotic centrosome separation / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / mitotic centrosome separation / COPI-mediated anterograde transport / COPI-dependent Golgi-to-ER retrograde traffic / kinesin complex / microtubule motor activity / spindle organization / microtubule-based movement / mitotic spindle assembly / MHC class II antigen presentation / mitotic spindle organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / mitotic spindle / spindle / microtubule cytoskeleton organization / spindle pole / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / microtubule / cell division / GTPase activity / GTP binding / protein kinase binding / protein-containing complex / ATP binding / membrane / metal ion binding / nucleus / cytosol / 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 beta chain / Kinesin-like protein KIF11 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human) / Pig (pig)
Methodhelical reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsPena AP / Sweeney A / Cook AD / Moores CA / Topf M
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Wellcome Trust202679/Z/16/Z and 206166/Z/17/Z United Kingdom
Medical Research Council (United Kingdom)MR/N013867/1 United Kingdom
Medical Research Council (United Kingdom)MR/J003867/1 United Kingdom
Worldwide Cancer Research
CitationJournal: Structure / Year: 2020
Title: Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy.
Authors: Alejandro Peña / Aaron Sweeney / Alexander D Cook / Julia Locke / Maya Topf / Carolyn A Moores /
Abstract: Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which ...Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.
History
DepositionOct 29, 2019-
Header (metadata) releaseDec 18, 2019-
Map releaseMar 4, 2020-
UpdateApr 15, 2020-
Current statusApr 15, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ta4
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10422.png

Downloads & links

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Map

FileDownload / File: emd_10422.map.gz / Format: CCP4 / Size: 2.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 0.0125 / Movie #1: 0.0125
Minimum - Maximum0 - 0.052762322
Average (Standard dev.)0.0023877716 (±0.006027386)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin16260143
Dimensions6686115
Spacing8666115
CellA: 119.54 Å / B: 91.74 Å / C: 159.84999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z8666115
origin x/y/z0.0000.0000.000
length x/y/z119.54091.740159.850
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS60162143
NC/NR/NS8666115
D min/max/mean0.0000.0530.002

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Supplemental data

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Sample components

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Entire : AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin

EntireName: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
Components
  • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
    • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
      • Protein or peptide: Tubulin beta chain
      • Protein or peptide: Tubulin alpha-1B chain
    • Complex: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
      • Protein or peptide: Kinesin-like protein KIF11
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin

SupramoleculeName: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin

SupramoleculeName: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin

SupramoleculeName: AMPPNP bound Human Eg5 motor domain in complex with alpha/beta tubulin
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig)
Molecular weightTheoretical: 48.113129 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 QDAT

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Macromolecule #2: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig)
Molecular weightTheoretical: 48.780117 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVD

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Macromolecule #3: Kinesin-like protein KIF11

MacromoleculeName: Kinesin-like protein KIF11 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.727461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHSSG VDLGTENLYF QSMASQPNSS AKKKEEKGKN IQVVVRCRPF NLAERKASAH SIVECDPVRK EVSVRTGGLA DKSSRKTYT FDMVFGASTK QIDVYRSVVC PILDEVIMGY NCTIFAYGQT GTGKTFTMEG ERSPNEEYTW EEDPLAGIIP R TLHQIFEK ...String:
MHHHHHHSSG VDLGTENLYF QSMASQPNSS AKKKEEKGKN IQVVVRCRPF NLAERKASAH SIVECDPVRK EVSVRTGGLA DKSSRKTYT FDMVFGASTK QIDVYRSVVC PILDEVIMGY NCTIFAYGQT GTGKTFTMEG ERSPNEEYTW EEDPLAGIIP R TLHQIFEK LTDNGTEFSV KVSLLEIYNE ELFDLLNPSS DVSERLQMFD DPRNKRGVII KGLEEITVHN KDEVYQILEK GA AKRTTAA TLMNAYSSRS HSVFSVTIHM KETTIDGEEL VKIGKLNLVD LAGSENIGRS GAVDKRAREA GNINQSLLTL GRV ITALVE RTPHVPYRES KLTRILQDSL GGRTRTSIIA TISPASLNLE ETLSTLEYAH RAKNILNKPE VNQKL

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Macromolecule #4: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / 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 / Guanosine triphosphate

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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 statefilament

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Sample preparation

BufferpH: 7.2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
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: 45.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.95 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.7 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 43714
FSC plot (resolution estimation)

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