|Entry||Database: EMDB / ID: 1032|
|Title||A new look at the microtubule binding patterns of dimeric kinesins.|
|Map data||rat kinesin dimer, modified at position 339 (A-C) which allowed to crosslink the dimer at the beginning of the neck helix|
|Sample||rat kinesin dimer:|
rat kinesin / tubulin
|Source||Rattus norvegicus (Norway rat)|
|Method||helical reconstruction / cryo EM / negative staining / 25 Å resolution|
|Citation||Journal: J. Mol. Biol. / Year: 2000|
Title: A new look at the microtubule binding patterns of dimeric kinesins.
Authors: A Hoenger / M Thormählen / R Diaz-Avalos / M Doerhoefer / K N Goldie / J Müller / E Mandelkow
Abstract: The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good ...The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.
|Date||Deposition: Feb 27, 2003 / Header (metadata) release: Feb 27, 2003 / Map release: Feb 27, 2003 / Last update: Apr 13, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_1032.map.gz (map file in CCP4 format, 3908 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 5.676 Å|
CCP4 map header:
-Entire rat kinesin dimer
|Entire||Name: rat kinesin dimer / Oligomeric State: dimer / Number of components: 2|
-Component #1: protein, rat kinesin
|Protein||Name: rat kinesin / a.k.a: molecular motor / Oligomeric Details: dimer / Number of Copies: 1 / Recombinant expression: Yes|
|Source||Species: Rattus norvegicus (Norway rat)|
-Component #2: protein, tubulin
|Protein||Name: tubulin / a.k.a: microtubulesMicrotubule / Oligomeric Details: hetero dimer / Recombinant expression: No / Number of Copies: 1|
|Source||Species: Rattus norvegicus (Norway rat)|
|Source (natural)||Location in cell: cytoplasm / Cell: neuronal cells / Organ or tissue: brain|
|Specimen||Specimen state: filament / Method: negative staining, cryo EM|
|Helical parameters||Axial symmetry: C1 (asymmetric) / Hand: LEFT HANDED|
|Sample solution||Specimen conc.: 0.5 mg/ml|
Buffer solution: Pipes 20mM, 50 mM NaCl, 5mM mgcl,1mM Mg-ATP, 20microl taxol.
|Support film||holey grids|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 93 K / Details: Vitrification instrument: self made|
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS CM120T|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 100 kV / Electron dose: 5 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 37000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2000 nm|
|Specimen Holder||Holder: side-entry / Model: GATAN LIQUID NITROGEN|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 14 / Scanner: ZEISS SCAI / Sampling size: 21 microns / Bit depth: 16|
|Processing||Method: helical reconstruction|
|3D reconstruction||Algorithm: helical / Software: Phoelix, Suprim / Resolution: 25 Å / Resolution method: FSC 0.5|
Details: Final maps from 28 averaged datasets = 14 helical tubes
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