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- EMDB-5417: Cryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexe... -

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Basic information

Entry
Database: EMDB / ID: EMD-5417
TitleCryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the AMP-PNP state (represents the ATP bound state)
Map dataReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state
Sample
  • Sample: GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
  • Protein or peptide: GCN4-Kar3Vik1
  • Protein or peptide: alpha-beta tubulin
KeywordsKar3Vik1 / kinesin-14 / microtubule / spindle stabilization in mitosis
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Bos taurus (domestic cattle)
Methodhelical reconstruction / cryo EM / Resolution: 24.5 Å
AuthorsCope J / Rank KC / Gilbert S / Rayment I / Hoenger A
CitationJournal: J Cell Biol / Year: 2012
Title: Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.
Authors: Katherine C Rank / Chun Ju Chen / Julia Cope / Ken Porche / Andreas Hoenger / Susan P Gilbert / Ivan Rayment /
Abstract: Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the ...Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.
History
DepositionMay 1, 2012-
Header (metadata) releaseMay 15, 2012-
Map releaseFeb 6, 2013-
UpdateMar 2, 2016-
Current statusMar 2, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5417.tif

Downloads & links

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Map

FileDownload / File: emd_5417.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 127 pix.
= 482.6 Å		3.8 Å/pix.
x 149 pix.
= 566.2 Å		3.8 Å/pix.
x 149 pix.
= 566.2 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: 3.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 8.0 / Movie #1: 8
Minimum - Maximum-52.58811188 - 69.826965329999993
Average (Standard dev.)1.28953886 (±14.577727319999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions149149127
Spacing149149127
CellA: 566.2 Å / B: 566.2 Å / C: 482.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z149149127
origin x/y/z0.0000.0000.000
length x/y/z566.200566.200482.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS149149127
D min/max/mean-52.58869.8271.290

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Supplemental data

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Sample components

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Entire : GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state

EntireName: GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
Components
  • Sample: GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
  • Protein or peptide: GCN4-Kar3Vik1
  • Protein or peptide: alpha-beta tubulin

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Supramolecule #1000: GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state

SupramoleculeName: GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
type: sample / ID: 1000
Details: GCN4-Kar3Vik1 was incubated with the non-hydrolyzable ATP analog AMP-PNP to trap the motor in the ATP-state conformation.
Oligomeric state: One heterodimer of Kar3Vik1 binds to one heterodimer of alpha-beta tubulin
Number unique components: 2

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Macromolecule #1: GCN4-Kar3Vik1

MacromoleculeName: GCN4-Kar3Vik1 / type: protein_or_peptide / ID: 1
Details: This truncated version of Kar3Vik1 contains the complete C-terminal globular domains as well as two and a half heptads of the native coiled coil. The GCN4 leucine zipper sequence was added ...Details: This truncated version of Kar3Vik1 contains the complete C-terminal globular domains as well as two and a half heptads of the native coiled coil. The GCN4 leucine zipper sequence was added to the N-terminus to initialize dimerization.
Number of copies: 1 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Spindle poles
Molecular weightExperimental: 87 KDa / Theoretical: 87 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24d (Kar3), pKLD37 (Vik1)

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Macromolecule #2: alpha-beta tubulin

MacromoleculeName: alpha-beta tubulin / type: protein_or_peptide / ID: 2 / Details: Heterodimer of alpha and beta tubulin / Number of copies: 1 / Oligomeric state: Heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: bovine / Tissue: brain / Location in cell: cytosol
Molecular weightExperimental: 110 KDa / Theoretical: 110 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.70 mg/mL
BufferpH: 7.2
Details: 20mM HEPES, 5mM magnesium acetate, 50mM potassium acetate, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT
GridDetails: C-flat 200 mesh copper grid with holey carbon film.
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification carried out at room temperature
Method: 5 uL of 0.41 mg/mL microtubules was adsorbed to a grid for 45 seconds. Excess liquid was blotted away and immediately 5 uL of 0.70 mg/mL Kar3Vik1 complexed with AMP-PNP was added to the ...Method: 5 uL of 0.41 mg/mL microtubules was adsorbed to a grid for 45 seconds. Excess liquid was blotted away and immediately 5 uL of 0.70 mg/mL Kar3Vik1 complexed with AMP-PNP was added to the microtubules for 2 minutes. Excess liquid was blotted for approximately 2.5 seconds prior to plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 95 K / Max: 97 K / Average: 96 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DetailsLow-dose cryo-EM recording
DateJun 8, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 34 / Average electron dose: 15 e/Å2 / Details: Recorded on CCD 4K camera / Od range: 1.4 / Bits/pixel: 14
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder: GATAN 626 cryo-holder / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsHelical processing was carried out with PHOELIX
Final reconstructionApplied symmetry - Helical parameters - Δz: 10.666 Å
Applied symmetry - Helical parameters - Δ&Phi: 24 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD, PHOELIX, SUPRIM
Details: Final map was calculated from an average of 67 datasets including approximately 27000 asymmetric units.

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