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- EMDB-8408: Cryo-EM reconstruction of the yeast kinesin-5, Cin8, bound to GDP... -

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Basic information

Entry
Database: EMDB / ID: 8408
TitleCryo-EM reconstruction of the yeast kinesin-5, Cin8, bound to GDP-taxol microtubules in ADP-AlFx state
Map data3D helical reconstruction of Cin8 motor domain in ADP-AlFx state bound to microtubule using cryo-EM
SampleMicrotubule-bound kinesin-5 Cin8 in ADP-AlFx state:
SourceSaccharomyces cerevisiae (baker's yeast)
Methodhelical reconstruction / cryo EM / 7.3 Å resolution
AuthorsCha HK
CitationJournal: J. Biol. Chem. / Year: 2017
Title: The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner.
Authors: Kayla M Bell / Hyo Keun Cha / Charles V Sindelar / Jared C Cochran
Abstract: Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in can move bidirectionally along ...Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single "canonical" site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate ("noncanonical") sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal.
DateDeposition: Oct 1, 2016 / Header (metadata) release: Oct 12, 2016 / Map release: Jul 26, 2017 / Last update: Sep 13, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0957
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0957
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0957
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8408.map.gz (map file in CCP4 format, 87809 KB)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
280 pix
2.49 Å/pix.
= 698.326 Å
280 pix
2.49 Å/pix.
= 698.326 Å
280 pix
2.49 Å/pix.
= 698.326 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.49402 Å
Density
Contour Level:0.0957 (by author), 0.0957 (movie #1):
Minimum - Maximum0 - 0.21301804
Average (Standard dev.)0.0074020033 (0.024142148)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions280280280
Origin-140-140-140
Limit139139139
Spacing280280280
CellA=B=C: 698.3256 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.49402142857142.49402142857142.4940214285714
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z698.326698.326698.326
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-140-140-140
NC/NR/NS280280280
D min/max/mean0.0000.2130.007

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Supplemental data

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Sample components

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Entire Microtubule-bound kinesin-5 Cin8 in ADP-AlFx state

EntireName: Microtubule-bound kinesin-5 Cin8 in ADP-AlFx state
Details: Cin8-his monomer (residues 70-535) Taxol-stabilized microtubules
Number of components: 1

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Component #1: protein, Microtubule-bound kinesin-5 Cin8 in ADP-AlFx state

ProteinName: Microtubule-bound kinesin-5 Cin8 in ADP-AlFx state
Details: Cin8-his monomer (residues 70-535) Taxol-stabilized microtubules
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET16b / Cell of expression system: B834(DE3)

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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: 9.455 Å / Delta phi: -25.77 deg.
Sample solutionBuffer solution: adjusted to pH 6.8 with KOH / pH: 6.8
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 1.71 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: FREALIGN / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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