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Cryo-electron microscopy of the kinesin-14 GCN4-Kar3Vik1 complexed to microtubules in the nucleotide-free state

by helical reconstruction, at 22 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 14, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 14, Made by UCSF CHIMERA

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 5416
AuthorsCope J, Rank KC, Gilbert S, Rayment I, Hoenger A
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 14, Made by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 14, Made by UCSF CHIMERA

Supplemental images

emd_5416.tif
Structure viewersYorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Related Entries

EMDB-5417
Cite

Cite: data citing same article

Similar strucutres (beta)

Diagram

EMDB-5417

EMDB-1702

EMDB-1612
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.
LinksDOI: 10.1371/journal.pone.0053792, PubMed: 23342004, PMC: PMC3544905
Citation - #1
ArticleJ. Cell Biol., Vol. 197, Issue 7, Page 957-70, Year 2012
TitleKar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.
AuthorsKatherine C Rank, Chun Ju Chen, Julia Cope, Ken Porche, Andreas Hoenger, Susan P Gilbert, Ivan Rayment
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
KeywordsAdenosine Diphosphate (metabolism, 58-64-0), Cryoelectron Microscopy, Fungal Proteins (chemistry), KAR3 protein, S cerevisiae, Kinesin (chemistry, 3.6.1.-), Microtubule-Associated Proteins (chemistry), Microtubules (metabolism), Models, Molecular, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Saccharomyces cerevisiae (chemistry), Saccharomyces cerevisiae Proteins (chemistry), VIK1 protein, S cerevisiae
LinksDOI: 10.1083/jcb.201201132, PubMed: 22734002, PMC: PMC3384419
Map
FileEMD-5416.map ( map file in CCP4 format, 11280 KB )
Projections & SlicesSize of images:
AxesZ (Sec.)Y (Row.)X (Col.)
Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Density

Histogram

Histogram (log scale)
Contour Level:14 (by author), 14 (movie #1):
Minimum - Maximum: -51.49787140 - 68.80082703
Average (Standard dev.): 1.28552449 (14.24060059)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis Order : X Y Z
Dimensions : 149 149 127
Origin : 0 0 0
Limit : 148 148 126
Spacing : 149 149 127
Unit CellA = 566.2 A , B = 566.2 A , C = 482.6 A ,
alpha = 90.0 degrees , beta = 90.0 degrees , gamma = 90.0 degrees
Pixel SpacingX = 3.8 A , Y = 3.8 A , 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-51.49868.8011.286
Annotation DetailsReconstruction of the kinesin-14 GCN4-Kar3Vik1 bound to microtubules in the nucleotide-free state
Supplement
Images
Images

emd_5416.tif
Sample
NameGCN4-Kar3Vik1 bound to microtubules in the nucleotide-free state
Number of Components2
Oligomeric StateOne heterodimer of Kar3Vik1 binds to one heterodimer of alpha-beta tubulin
DetailsGCN4-Kar3Vik1 was treated with the ATP/ADP hydrolyzing enzyme Apyrase prior to incubation with microtubules to generate the nucleotide-free-state microtubule-bound motor 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 (NCBI Taxonomy: 4932)

Common Name of SpeciesBaker's yeast
Recombinant expressionYes
Natural SourceCell Location: Spindle poles
Engineered SourceExp System: Escherichia coli (NCBI Taxonomy: 562)
Vector: pET24d (Kar3), pKLD37 (Vik1)
Component #2: protein - alpha-beta tubulin
Scientific namealpha-beta tubulin
Theoretical Mass0.110 MDa
Experimental Mass0.110 MDa
DetailsHeterodimer of alpha and beta tubulin
Oligomeric DetailsHeterodimer
Number of Copies1
Scientific Name of SpeciesBos taurus (NCBI Taxonomy: 9913)

Common Name of Speciesbovine
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 Conc0.39 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; 5 uL of 0.39 mg/mL apyrase-treated Kar3Vik1 was immediately 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
Date06-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 X,
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 ( GATAN LIQUID NITROGEN )
Temperature96 Kelvin ( 95 Kelvin - 97 Kelvin )
Camera
DetectorGatan Ultrascan 895 CCD camera
Image Acquisition
Od Range1.4
Number of Digital Images26
Sampling Size15 microns
Quant Bit Number14
DetailsRecorded on CCD 4K camera
Processing
Methodhelical reconstruction
3 D reconstruction
AlgorithmHelical reconstruction using PHOELIX
SoftwareIMOD, PHOELIX, SUPRIM
DetailsFinal map was calculated from an average of 52 datasets including approximately 42000 asymmetric units.
Resolution By Author22
Resolution MethodFSC at 0.5 cut-off
Helical
DetailsHelical processing was carried out with PHOELIX
Download
Data from EMDB
Header (meta data in XML format)emd-5416.xml (10.7 KB)
Map dataemd_5416.map.gz (10.1 MB)
Imagesemd_5416.tif (418.2 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-5416
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.3 MB
.webm (WebM/VP8 format), 5.3 MB
Session file for UCSF-Chimera, 27 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.1 MB
.webm (WebM/VP8 format), 4.8 MB
Session file for UCSF-Chimera, 27.1 KB