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- EMDB-2077: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -

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Basic information

Entry
Database: EMDB / ID: EMD-2077
TitleElectron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in AMPPNP state.
Map data3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site
Sample
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
  • Protein or peptide: alpha-beta tubulin dimer
  • Protein or peptide: Kinesin-5 motor domain
Keywordscancer / electron cryo-microscopy / kinesin / microtubule / mitosis
Function / homology
Function and homology information


spindle elongation / Kinesins / plus-end-directed microtubule motor activity / regulation of mitotic centrosome separation / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / kinesin complex / microtubule motor activity / spindle organization ...spindle elongation / Kinesins / plus-end-directed microtubule motor activity / regulation of mitotic centrosome separation / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / kinesin complex / microtubule motor activity / spindle organization / microtubule-based movement / mitotic spindle assembly / microtubule-based process / 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 / spindle / microtubule cytoskeleton organization / spindle pole / mitotic spindle / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / 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
Kinesin-like protein KIF11 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsGoulet A / Behnke-Parks WM / Sindelar C / Rosenfeld S / Moores C
CitationJournal: J Biol Chem / Year: 2012
Title: The structural basis of force generation by the mitotic motor kinesin-5.
Authors: Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved ...Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
History
DepositionApr 19, 2012-
Header (metadata) releaseMay 17, 2012-
Map releaseNov 21, 2012-
UpdateJan 9, 2013-
Current statusJan 9, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4aqv
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4aqv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2077.map.gz / Format: CCP4 / Size: 348.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-6.57703495 - 8.06319046
Average (Standard dev.)0.08032296 (±1.81039548)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions454545
Spacing454545
CellA=B=C: 126.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z454545
origin x/y/z0.0000.0000.000
length x/y/z126.000126.000126.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS454545
D min/max/mean-6.5778.0630.080

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Supplemental data

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Sample components

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Entire : 13-protofilament microtubule-bound human kinesin-5 motor domain w...

EntireName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
Components
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
  • Protein or peptide: alpha-beta tubulin dimer
  • Protein or peptide: Kinesin-5 motor domain

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Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...

SupramoleculeName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
type: sample / ID: 1000
Oligomeric state: 13-protofilament microtubule with one kineisn-5 motor domain bound every tubulin heterodimers
Number unique components: 2

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Macromolecule #1: alpha-beta tubulin dimer

MacromoleculeName: alpha-beta tubulin dimer / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain

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Macromolecule #2: Kinesin-5 motor domain

MacromoleculeName: Kinesin-5 motor domain / type: protein_or_peptide / ID: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pet21a

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5mM AMPPNP
GridDetails: 400 mesh holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: chamber at 24 degrees C, blot 2.5 sec

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateJan 10, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 46 / Average electron dose: 18 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: FREALIGN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Approximately 50,000 asymmetric units were averaged in the final reconstruction.
Number images used: 3587
DetailsThe particles were selected along individual microtubules.

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera, FlexEM
DetailsProtocol: rigid body then flexible fitting. The domain was fitted as a rigid body. The N-terminal residues 6 to 16 were built in the EM map and the final model was refined by flexible fitting.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation
Output model

PDB-4aqv:
Model of human kinesin-5 motor domain (3HQD) and mammalian tubulin heterodimer (1JFF) docked into the 9.7-angstrom cryo-EM map of microtubule-bound kinesin-5 motor domain in the AMPPPNP state.

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Atomic model buiding 2

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsProtocol: rigid body. alpha- and b-tubulin were separately fitted.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-4aqv:
Model of human kinesin-5 motor domain (3HQD) and mammalian tubulin heterodimer (1JFF) docked into the 9.7-angstrom cryo-EM map of microtubule-bound kinesin-5 motor domain in the AMPPPNP state.

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