The Boulder Laboratory for 3-D Microscopy of Cells, University of Colorado at Boulder, Department of Molecular, Cellular, and Developmental Biology, Boulder, CO 80309-0347, USA.
J Struct Biol. 2010 May;170(2):257-65. doi: 10.1016/j.jsb.2009.12.004. Epub 2009 Dec 16.
Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods.
多年来，与驱动蛋白家族的分子马达或非马达微管相关蛋白（MAPs）如 tau 或 EB1 复合的微管一直是基于冷冻电子显微镜的 3D 研究的主题。这些针对完整微管复合物的研究大多数是通过螺旋 3D 重建进行的，而少数是通过单颗粒方法或二维结晶阵列进行分析的。微管-MAP 或马达复合物的螺旋重建已经非常成功，但从定义上讲，所有螺旋 3D 重建尝试都需要完美的螺旋组装，这是一个严重的限制，并且将尝试限制在 15 或 16 原丝微管，这种微管结构在自然界中非常罕见。在过去几年中，冷冻电子断层摄影术的兴起为解决不形成螺旋组装的微管-MAP 复合物的 3D 结构开辟了一条新途径，对于这里的主题来说，最重要的是，所有表现出晶格缝的微管。此外，并非所有的马达结构域或 MAPs 都能以足够的规则来修饰微管表面，以匹配底层微管晶格，或者它们采用偏离螺旋对称的构象。在这里，我们使用两个驱动蛋白结构域，单体 Eg5 驱动蛋白结构域和异源二聚体 Kar3Vik1 驱动蛋白结构域，展示了冷冻电子断层摄影术的强大功能和局限性。我们在这里表明，当可以从断层图像中提取相同的子体积时，断层摄影术并不排除后断层摄影术平均的可能性，并且在这两种情况下，我们都能够重建无法使用螺旋或其他基于平均的方法获得的构象的 3D 图谱。
