CRYOET OF WT & MUTANT FLAGELLA OF CHLAMYDOMONAS ALLOWS MAPPING OF IDA SUBUNITS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Axonemal dynein是一种大型的微管马达酶，它将ATP结合和水解产生的化学能转化为弯曲纤毛和鞭毛的机械力。虽然对鞭毛机制的某些方面的了解已经取得了很大的进展，但对于单个动力蛋白亚基的功能，它们在轴丝S 96 nm轴向重复序列中的结构组织，以及马达与协调其节拍模式的调节机制之间的关系，仍有许多有待了解。因此，我们正在使用冷冻电子断层扫描和相关平均(参见子项目T0066)相结合的方法来研究野生型和突变型鞭毛的三维结构，以提高断层重建的信噪比，从而提高其分辨率。我们已经记录了从野生型衣藻和不同的内动力蛋白臂突变株(PF9、6F5、PF10)分离的几个倾斜系列的去膜和快速冷冻的轴丝。衣藻野生型轴丝96 nm重复序列的三维重建和体积平均值证实了内外动力蛋白臂复合体的主要结构特征。然而，与以前结构研究提供的二维图像相比，断层扫描体积允许进行三维分析，并揭示了新的结构细节，例如内外动力蛋白臂复合体之间的两个新的连接子结构。我们正在使用平均野生型和突变型轴丝的三维差异图来原位确定特定多肽缺陷引起的结构变化。

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. Axonemal dyneins are large, microtubule-based motor enzymes that convert the chemical energy derived from ATP binding and hydrolysis into the mechanical forces that bend cilia and flagella. While great progress has been made in understanding some aspects of flagellar mechanisms, much remains to be learned about the function of individual dynein subunits, their structural organization within the axoneme¿s 96 nm axial repeat, and the relationship between the motors and the regulatory machinery that coordinates their pattern of beat. We are therefore studying the 3-D structure of wild-type and mutant flagella using cryo-electron tomography in combination with correlation averaging (see Subproject T0066) to improve the signal-to-noise ratio in the tomographic reconstructions, and thus to enhance their resolution. We have recorded several tilt series of demembranated and rapidly frozen axonemes isolated from wild-type Chlamydomonas and from different inner dynein arm mutant strains (pf9, 6F5, pf10). The 3-D reconstructions and volume averages of the 96nm repeat of Chlamydomonas wild type axonemes confirm the main structural features of the inner and outer dynein arm complexes. However, in comparison to the 2-D pictures provided by previous structural studies, the tomographic volumes allow a 3D analysis and reveal new structural details, such as two novel linker structures between the inner and outer dynein arm complexes. We are using 3-D difference maps of averaged wild type and mutant axonemes to determine in situ the structural alterations that result from specific polypeptide defects.