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Cryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the AMP-PNP state (represents the ATP bound state)

by helical reconstruction, at 24.5 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 8, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 8, Made by UCSF CHIMERA

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 5417
TitleCryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the AMP-PNP state (represents the ATP bound state)
MapReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state
SampleGCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
KeywordsKar3Vik1, kinesin-14, microtubule, spindle stabilization in mitosis
AuthorsCope J, Rank KC, Gilbert S, Rayment I, Hoenger A
DateDeposition: 2012-05-01, Header release: 2012-05-15, Map release: 2013-02-06, Last update: 2013-02-06
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 8, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 8, Made by UCSF CHIMERA

Supplemental images
Structure viewersYorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Related Entries

Cite: data citing same article

Similar strucutres (beta)
List of similar structure data about Omokage system
Article
Citation - Primary
ArticlePLoS ONE, Vol. 8, Issue 1, Page e53792, Year 2013
TitleKar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.
AuthorsJulia Cope, Katherine C Rank, Susan P Gilbert, Ivan Rayment, Andreas Hoenger
The Boulder Laboratory for 3-D Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
KeywordsAdenosine Diphosphate (metabolism), Adenosine Triphosphate (metabolism), Amino Acid Sequence, Animals, Cattle, Cryoelectron Microscopy, Kinesin (chemistry, 3.6.1.-), Microtubules (metabolism), Models, Molecular, Molecular Sequence Data, Movement, Phosphates (metabolism), Protein Conformation, Protein Multimerization, Rotation, Stochastic Processes
LinksDOI: 10.1371/journal.pone.0053792, PubMed: 23342004, PMC: PMC3544905
Citation - #1
ArticleJ.CELL BIOL., Vol. 197, Page 957-970, Year 2012
TitleKar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.
AuthorsRank KC, Chen CJ, Cope J, Porche K, Hoenger A, Gilbert SP, Rayment I
Map
Fileemd_5417.map.gz ( map file in CCP4 format, 11015 KB )
Projections & SlicesSize of images:
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.

Density
Contour Level:8 (by author), 8 (movie #1):
Minimum - Maximum: -52.58811188 - 69.82696533
Average (Standard dev.): 1.28953886 (14.57772732)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions149149127
Origin000
Limit148148126
Spacing149149127
Unit CellA: 566.2 A, B: 566.2 A, C: 482.6 A
Alpha=beta=gamma: 90 degrees
Pixel SpacingX= Y= Z: 3.8 A
CCP4 map header info
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-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS149149127
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean-52.58869.8271.290
Annotation DetailsReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state, representative of the ATP-bound state
Supplement
Images
Images
Sample
NameGCN4-Kar3Vik1 bound to microtubules in the AMP-PNP state
Number of Components2
Oligomeric StateOne heterodimer of Kar3Vik1 binds to one heterodimer of alpha-beta tubulin
DetailsGCN4-Kar3Vik1 was incubated with the non-hydrolyzable ATP analog AMP-PNP to trap the motor in the ATP-state conformation.
Component #1: protein - GCN4-Kar3Vik1
Scientific nameGCN4-Kar3Vik1
Theoretical Mass0.087 MDa
Experimental Mass0.087 MDa
DetailsThis 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.
Oligomeric DetailsHeterodimer
Number of Copies1
Scientific Name of SpeciesSaccharomyces cerevisiae

Common Name of SpeciesBaker's yeast
NCBI taxonomy4932
Recombinant expressionYes
Natural SourceCell Location: Spindle poles
Engineered SourceNCBI taxonomy: 562
Expression system: Escherichia coli
Vector: pET24d (Kar3), pKLD37 (Vik1)
Component #2: protein - alpha-beta tubulin
Scientific namealpha-beta tubulin
Theoretical Mass0.11 MDa
Experimental Mass0.11 MDa
DetailsHeterodimer of alpha and beta tubulin
Oligomeric DetailsHeterodimer
Number of Copies1
Scientific Name of SpeciesBos taurus

Common Name of Speciesbovine
NCBI taxonomy9913
Recombinant expressionNo
Natural SourceCell Location: cytosol
Organ Or Tissue: brain
Experiment
Sample Preparation
Helical ParametersAxial Symmetry: s1
Delta Z: 10.666 A
Delta Phi: 24 degrees
Hand: LEFT HANDED
Specimen Conc0.7 mg/ml
Specimen Support DetailsC-flat 200 mesh copper grid with holey carbon film.
Specimen Statefilament
BufferpH: 7.2
Details: 20mM HEPES, 5mM magnesium acetate, 50mM potassium acetate, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT
Vitrification
Method5 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.
Cryogen NameETHANE
DetailsVitrification carried out at room temperature
InstrumentHOMEMADE PLUNGER
Temperature93 Kelvin
Imaging
MicroscopeFEI TECNAI F20
Date08-JUN-2011
DetailsLow-dose cryo-EM recording
Electron Gun
Electron SourceFIELD EMISSION GUN
Accelerating Voltage200 kV
Electron Dose15 e/A**2
Illumination ModeFLOOD BEAM
Lens
MagnificationNominal: 29000
AstigmatismObjective lens astigmatism was corrected at 100,000 times magnification.
Nominal Cs2 mm
Imaging ModeBRIGHT FIELD
Defocus1500 nm - 2500 nm
Specimen Holder
HolderGATAN 626 cryo-holder
ModelGATAN LIQUID NITROGEN
Temperature96 K ( 95 - 97 K)
Camera
DetectorGATAN ULTRASCAN 4000 (4k x 4k)
Image Acquisition
Od Range1.4
Number of Digital Images34
Sampling Size15
Quant Bit Number14
DetailsRecorded on CCD 4K camera
Processing
Methodhelical reconstruction
3D reconstruction
AlgorithmHelical reconstruction using PHOELIX
SoftwareIMOD, PHOELIX, SUPRIM
DetailsFinal map was calculated from an average of 67 datasets including approximately 27000 asymmetric units.
Resolution By Author24.5 A
Resolution MethodFSC 0.5
Helical
DetailsHelical processing was carried out with PHOELIX
Download
Data from EMDB
Header (meta data in XML format)emd-5417.xml (10.6 KB)
Map dataemd_5417.map.gz (10.1 MB)
Imagesemd_5417.tif (407.5 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-5417
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.4 MB
.webm (WebM/VP8 format), 5.4 MB
Session file for UCSF-Chimera, 27 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.2 MB
.webm (WebM/VP8 format), 4.9 MB
Session file for UCSF-Chimera, 27.1 KB