STRUCTURAL BASIS FOR MICROTUBULE NUCLEATION BY THE GAMMA-TUBULIN SMALL COMPLEX

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 中心体在微管骨架的组织中起着重要的作用。 中心体功能缺陷导致染色体分离错误，这是癌症发生和发展的主要因素。 我们的目标是了解微管成核在中心体的机制和调节，通过专注于一个关键的300 kDa的异源四聚体复合物的结构，在所有真核生物中的成核基础：<$-微管蛋白小复合物（<$-TuSC）。 我们已经确定了一个负应变EM结构的随机锥形倾斜方法，并在低温条件下的初步图像。从更高分辨率的cryo-EM结构中，我们希望确定复合物的整体组织，特别是在微管负端与<$$>-微管蛋白相互作用的<$-微管蛋白的表面，并绘制连接<$-TuSC与微管组织中心的蛋白质的结合位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The centrosome plays a fundamental role in organizing the microtubule cytoskeleton. Defects in centrosome function lead to errors in chromosome segregation, a major factor in both the initiation and progression of cancer. We aim to understand the mechanism and regulation of microtubule nucleation at the centrosome by focusing on the structure of a key 300 kDa heterotetrameric complex that underlies nucleation in all eukaryotes: the ¿-tubulin small complex (¿-TuSC). We have determined a negative stain EM structure by the random conical tilt method, and have preliminary images under cryo conditions. From a higher resolution cryo-EM structure we hope to determine the overall organization of the complex and particularly which surfaces of ¿-tubulin are exposed for interaction with ¿¿-tubulin at the microtubule minus ends, and to map the binding sites of proteins which link ¿-TuSC to microtubule organizing centers.