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- EMDB-1604: 9 Angstrom Structure of an AMPPNP Microtubule-Kinesin5 Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-1604
Title9 Angstrom Structure of an AMPPNP Microtubule-Kinesin5 Complex
Map dataMicrotubule-kinesin5 complex
Sample
  • Sample: Microtubule-KLP61F complex with AMPPNP
  • Protein or peptide: Microtubule
  • Protein or peptide: KLP61F
Keywordsmicrotubule / tubulin / kinesin / kinesin-5 / KLP61F / Eg5 / mitosis
Function / homology
Function and homology information


aster / plus-end directed microtubule sliding / fusome organization / fusome / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centrosome separation / spindle elongation / mitotic spindle microtubule / microtubule bundle formation ...aster / plus-end directed microtubule sliding / fusome organization / fusome / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / centrosome separation / spindle elongation / mitotic spindle microtubule / microtubule bundle formation / plus-end-directed microtubule motor activity / mitotic centrosome separation / positive regulation of axon guidance / microtubule associated complex / kinesin complex / positive regulation of Golgi to plasma membrane protein transport / microtubule-based movement / Golgi organization / mitotic spindle pole / mitotic spindle assembly / cytoskeletal motor activity / protein secretion / microtubule-based process / mitotic spindle organization / spindle microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / spindle / microtubule cytoskeleton organization / mitotic spindle / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / Golgi apparatus / endoplasmic reticulum / ATP binding / metal ion binding / nucleus / 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 alpha-1A chain / Tubulin beta chain / Kinesin-like protein Klp61F / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle) / Drosophila melanogaster (fruit fly)
Methodhelical reconstruction / cryo EM / Resolution: 9.4 Å
AuthorsBodey AJ / Kikkawa M / Moores CA
CitationJournal: J Mol Biol / Year: 2009
Title: 9-Angström structure of a microtubule-bound mitotic motor.
Authors: Andrew J Bodey / Masahide Kikkawa / Carolyn A Moores /
Abstract: Kinesin-5 (K5) motors are important components of the microtubule (MT)-based cell division machinery and are targets for small-molecule inhibitors currently in cancer clinical trials. However, the ...Kinesin-5 (K5) motors are important components of the microtubule (MT)-based cell division machinery and are targets for small-molecule inhibitors currently in cancer clinical trials. However, the nature of the K5-MT interaction and the regulatory mechanisms that control it remain unclear. Using cryo-electron microscopy and image processing, we calculated the structure of a K5 motor bound to MTs at 9 A resolution, providing insight into this important interaction. Our reconstruction reveals the K5 motor domain in an ATP-like conformation in which MT binding induces the conserved nucleotide-sensing switch I and II loops to form a compact subdomain around the bound nucleotide. Our reconstruction also reveals a novel conformation for the K5-specific drug-binding loop 5, suggesting a possible role for it in switching K5s between force generation and diffusional modes of MT binding. Our data thus shed light on regulation of the interaction between spindle components important for chromosome segregation.
History
DepositionFeb 26, 2009-
Header (metadata) releaseMar 9, 2009-
Map releaseApr 1, 2009-
UpdateMar 13, 2013-
Current statusMar 13, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2wbe
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2wbe
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2wbe
  • 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_1604.map.gz / Format: CCP4 / Size: 12.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicrotubule-kinesin5 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 228 pix.
= 319.2 Å
1.4 Å/pix.
x 95 pix.
= 133. Å
1.4 Å/pix.
x 151 pix.
= 211.4 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: 1.4 Å
Density
Contour Level1: 0.5 / Movie #1: 0.5
Minimum - Maximum-3.80928 - 5.50361
Average (Standard dev.)-0.00000000110627 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-47-75-114
Dimensions95151228
Spacing95151228
CellA: 211.4 Å / B: 133 Å / C: 319.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z15195228
origin x/y/z0.0000.0000.000
length x/y/z211.400133.000319.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS-75-47-114
NC/NR/NS15195228
D min/max/mean-3.8095.504-0.000

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Supplemental data

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Sample components

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Entire : Microtubule-KLP61F complex with AMPPNP

EntireName: Microtubule-KLP61F complex with AMPPNP
Components
  • Sample: Microtubule-KLP61F complex with AMPPNP
  • Protein or peptide: Microtubule
  • Protein or peptide: KLP61F

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Supramolecule #1000: Microtubule-KLP61F complex with AMPPNP

SupramoleculeName: Microtubule-KLP61F complex with AMPPNP / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: Microtubule

MacromoleculeName: Microtubule / type: protein_or_peptide / ID: 1 / Name.synonym: Microtubule / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: cattle / Tissue: Brain

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Macromolecule #2: KLP61F

MacromoleculeName: KLP61F / type: protein_or_peptide / ID: 2 / Name.synonym: KLP61F / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightTheoretical: 42 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-15b-TEV

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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: 6.8
Details: 80mM PIPES, 150mM NaCl, 7mM MgCl2, 1mM EGTA, 1mM beta-mercaptoethanol
GridDetails: Quantifoil grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home made / Method: Blot for 2-3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.94 µm / Nominal defocus min: 1.08 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt cryo / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at 150,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7.0 µm / Number real images: 24 / Average electron dose: 10 e/Å2 / Bits/pixel: 12
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping, Wiener
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Ruby-Helix

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: UCSF Chimera
DetailsProtocol: Rigid body. KLP61F, alpha-tubulin and beta-tubulin were all fitted separately
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2wbe:
Kinesin-5-Tubulin Complex with AMPPNP

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: UCSF Chimera
DetailsProtocol: Rigid body. KLP61F, alpha-tubulin and beta-tubulin were all fitted separately
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2wbe:
Kinesin-5-Tubulin Complex with AMPPNP

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Atomic model buiding 3

Initial modelPDB ID:
SoftwareName: UCSF Chimera
DetailsProtocol: Rigid body. KLP61F, alpha-tubulin and beta-tubulin were all fitted separately
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2wbe:
Kinesin-5-Tubulin Complex with AMPPNP

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