|Entry||Database: EMDB / ID: 5417|
|Title||Cryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the AMP-PNP state (represents the ATP bound state)|
|Keywords||Kar3Vik1 / kinesin-14 / microtubule / spindle stabilization in mitosis|
|Sample||GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state|
|Source||Saccharomyces cerevisiae / yeast / Baker's yeast / サッカロミセス・セレビシエ / |
Bos taurus / mammal / bovine / ウシ /
|Map data||Reconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state|
|Method||helical reconstruction, at 24.5 A resolution|
|Authors||Cope J / Rank KC / Gilbert S / Rayment I / Hoenger A|
|Citation||PLoS ONE, 2013, 8, e53792-e53792|
primary. PLoS ONE, 2013, 8, e53792-e53792 StrPapers
1. J. Cell Biol., 2012, 197, 957-970 StrPapers
|Date||Deposition: May 1, 2012 / Header (metadata) release: May 15, 2012 / Map release: Feb 6, 2013 / Last update: May 1, 2012|
Downloads & links
|File||emd_5417.map.gz (map file in CCP4 format, 11015 KB)|
|Projections & slices||Size of images: |
Images are generated by Spider package.
|Voxel size||X=Y=Z: 3.8 A|
CCP4 map header:
-Entire GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
|Entire||Name: 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
-Component #1: protein, GCN4-Kar3Vik1
|Protein||Name: 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
|Mass||Theoretical: 87 kDa / Experimental: 87 kDa|
|Source||Species: 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|
-Component #2: protein, alpha-beta tubulin
|Protein||Name: alpha-beta tubulin / Oligomeric Details: Heterodimer / Details: Heterodimer of alpha and beta tubulin / Recombinant expression: No / Number of Copies: 1|
|Mass||Theoretical: 110 kDa / Experimental: 110 kDa|
|Source||Species: Bos taurus / mammal / bovine / ウシ /|
|Source (natural)||Location in cell: cytosol / Organ or tissue: brain|
|Helical parameters||Axial symmetry: C1 (asymmetric) / Hand: LEFT HANDED / Delta z: 10.666 A / Delta phi: 24 deg.|
|Sample solution||Specimen conc.: 0.7 mg/ml|
Buffer solution: 20mM HEPES, 5mM magnesium acetate, 50mM potassium acetate, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT
|Support film||C-flat 200 mesh copper grid with holey carbon film.|
|Vitrification||Instrument: 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
-Electron microscopy imaging
|Imaging||Microscope: FEI TECNAI F20 / Date: Jun 8, 2011 / Details: Low-dose cryo-EM recording|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 15 e/A2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Holder: GATAN 626 cryo-holder / Model: GATAN LIQUID NITROGEN / Temperature: 96 K ( 95 - 97 K)|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 34 / Sampling size: 15 microns / Bit depth: 14 / OD range: 1.4 / Details: Recorded on CCD 4K camera|
|Processing||Method: helical reconstruction / Details: Helical processing was carried out with PHOELIX|
|3D reconstruction||Algorithm: 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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