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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: 5417
TitleCryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the AMP-PNP state (represents the ATP bound state)
KeywordsKar3Vik1 / kinesin-14 / microtubule / spindle stabilization in mitosis
SampleGCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
SourceSaccharomyces cerevisiae / yeast / Baker's yeast / サッカロミセス・セレビシエ /
Bos taurus / mammal / bovine / ウシ /
Map dataReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state
Methodhelical reconstruction, at 24.5 A resolution
AuthorsCope J / Rank KC / Gilbert S / Rayment I / Hoenger A
CitationPLoS ONE, 2013, 8, e53792-e53792

primary. PLoS ONE, 2013, 8, e53792-e53792 StrPapers
Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.
Julia Cope / Katherine C Rank / Susan P Gilbert / Ivan Rayment / Andreas Hoenger

1. J. Cell Biol., 2012, 197, 957-970 StrPapers
Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.
Katherine C Rank / Chun Ju Chen / Julia Cope / Ken Porche / Andreas Hoenger / Susan P Gilbert / Ivan Rayment

DateDeposition: May 1, 2012 / Header (metadata) release: May 15, 2012 / Map release: Feb 6, 2013 / Last update: May 1, 2012

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 8
  • Imaged by UCSF CHIMERA
  • Download
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Supplemental images

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Map

Fileemd_5417.map.gz (map file in CCP4 format, 11015 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
127 pix
3.8 A/pix
= 482.6 A
149 pix
3.8 A/pix
= 566.2 A
149 pix
3.8 A/pix
= 566.2 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 3.8 A
Density
Contour Level:8 (by author), 8 (movie #1):
Minimum - Maximum-52.58811188 - 69.82696533
Average (Standard dev.)1.28953886 (14.57772732)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions149149127
Origin000
Limit148148126
Spacing149149127
CellA: 566.2 A / B: 566.2 A / C: 482.6 A
Alpha=beta=gamma: 90 deg.

CCP4 map header:

modeImage stored as Reals
A/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
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
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
Details: GCN4-Kar3Vik1 was incubated with the non-hydrolyzable ATP analog AMP-PNP to trap the motor in the ATP-state conformation.
Number of components: 2
Oligomeric State: One heterodimer of Kar3Vik1 binds to one heterodimer of alpha-beta tubulin

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Component #1: protein, GCN4-Kar3Vik1

ProteinName: GCN4-Kar3Vik1 / Oligomeric Details: Heterodimer
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 / Recombinant expression: Yes
MassTheoretical: 87 kDa / Experimental: 87 kDa
SourceSpecies: Saccharomyces cerevisiae / yeast / Baker's yeast / サッカロミセス・セレビシエ /
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Vector: pET24d (Kar3), pKLD37 (Vik1)
Source (natural)Location in cell: Spindle poles

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Component #2: protein, alpha-beta tubulin

ProteinName: alpha-beta tubulin / Oligomeric Details: Heterodimer / Details: Heterodimer of alpha and beta tubulin / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 110 kDa / Experimental: 110 kDa
SourceSpecies: Bos taurus / mammal / bovine / ウシ /
Source (natural)Location in cell: cytosol / Organ or tissue: brain

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Experimental details

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Sample preparation

Specimen statefilament
Helical parametersAxial symmetry: C1 (asymmetric) / Hand: LEFT HANDED / Delta z: 10.666 A / Delta phi: 24 deg.
Sample solutionSpecimen conc.: 0.7 mg/ml
Buffer solution: 20mM HEPES, 5mM magnesium acetate, 50mM potassium acetate, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT
pH: 7.2
Support filmC-flat 200 mesh copper grid with holey carbon film.
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 93 K
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.
Details: Vitrification carried out at room temperature

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI F20 / Date: Jun 8, 2011 / Details: Low-dose cryo-EM recording
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 15 e/A2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2500 nm
Specimen HolderHolder: GATAN 626 cryo-holder / Model: GATAN LIQUID NITROGEN / Temperature: 96 K ( 95 - 97 K)
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 34 / Sampling size: 15 microns / Bit depth: 14 / OD range: 1.4 / Details: Recorded on CCD 4K camera

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Image processing

ProcessingMethod: helical reconstruction / Details: Helical processing was carried out with PHOELIX
3D reconstructionAlgorithm: Helical reconstruction using PHOELIX / Software: IMOD, PHOELIX, SUPRIM
Details: Final map was calculated from an average of 67 datasets including approximately 27000 asymmetric units.
Resolution: 24.5 A / Resolution method: FSC 0.5

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