Structural investigations into microtubule-MAP complexes.
Microtubules interact with a large variety of factors commonly referred to as either molecular motors (kinesins, dyneins) or structural microtubule-associated proteins (MAPs). MAPs do not exhibit motor activity, but regulate microtubule dynamics and their interactions with molecular motors, and organelles such as kinetochores or centrosomes. Structural investigations into microtubule-kinesin motor complexes are quite advanced today and by helical three-dimensional (3-D) analysis reveal a resolution of the motor-tubulin interface at <1.0 nm. However, due to their flexible structure MAPs like tau or MAP2C cannot be visualized in the same straightforward manner. Helical averaging usually reveals only the location of strong binding sites while the overall structure of the MAP remains unsolved. Other MAPs such as EB1 bind very selectively only to some parts of the microtubule lattice such as the lattice seam. Thus, they do not reveal a stoichiometric tubulin:MAP-binding ratio that would allow for a quantitative helical 3-D analysis. Therefore, to get a better view on the structure of microtubule-MAP complexes we often used a strategy that combined cryo-electron microscopy and helical or tomographic 3-D analysis with freeze-drying and high-resolution unidirectional surface shadowing. 3-D analysis of ice-embedded specimens reveals their full 3-D volume. This relies either on a repetitive structure following a helical symmetry that can be used for averaging or suffers from the limited resolution that is currently achievable with cryotomography. Surface metal shadowing exclusively images surface-exposed features at very high contrast, adding highly valuable information to 2-D or 3-D data of vitrified structures.
Hoenger
Andreas
A
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, UCB-347, Boulder, Colorado 80309, USA.
Gross
Heinz
H
D020285:Cryoelectron Microscopy; D016547:Kinesins / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D012568:Schizosaccharomyces / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000648:ultrastructure; D016875:tau Proteins / Q000737:chemistry / Q000648:ultrastructure
D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; D016875:tau Proteins; D016547:Kinesins