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

WT 的秘密

• 批准号: 7355011
• 负责人: DANIELA NICASTRO
• 金额: $ 0.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355011
• 关键词: Chlamydomonas; arm; dynein ATPase; flagellum; mutant

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。轴突动力蛋白是大型的基于微管的运动酶，可将ATP结合和水解衍生的化学能转化为弯曲纤毛和鞭毛的机械力。尽管在理解鞭毛机制的某些方面取得了巨大的进步，但仍有许多待了解单个动力蛋白亚基的功能，其在轴突96 nm轴向重复轴上的结构组织以及电动机与调节机械之间的关系，这些组织可以协调其模式的模式。因此，我们正在研究使用冷冻电子断层扫描与相关平均（请参阅subproject t0066）结合使用野生型和突变鞭毛的3-D结构，以提高层析成像重建中的信噪比，从而提高其分辨率。我们记录了从野生型衣原体和不同内部动力蛋白臂突变菌株中分离出的几个倾斜的非植物和快速冷冻的轴突（PF9、6F5，PF10）。衣原体野生型轴突的96nm重复重复的3-D重建和体积平均值证实了内和外动力蛋白臂复合物的主要结构特征。但是，与以前的结构研究提供的2-D图片相比，层析成像量允许3D分析并揭示新的结构细节，例如内部和外动力蛋白臂复合物之间的两个新型接头结构。我们使用平均野生型和突变轴突的3-D差图来确定特定多肽缺陷导致的结构改变。