IMPROVEMENT OF TIME-RESOLVED INSTRUMENTATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要 时间分辨冷冻电子显微镜研究的目标是表征在大分子作用过程中出现的中间产物的结构。仪器、标本制备和数据分析方面的进展使用了现有内部项目的样本，这些项目受益于时间分辨成像：翻译核糖体的中间状态、钙释放通道的兰尼定受体类的功能状态(开放、关闭、失活/适应)以及线粒体的代谢状态。 对于这里感兴趣的大分子的大型复杂组装，其中一些中间产物预计涉及整个结构域或亚单位的移动，以及组成亚单位(例如，在复合物的组装或拆解中)或大分子配体(例如，与核糖体相互作用的tRNA)的损失或获得。这些类型的结构变化应该可以在通常由单粒子低温电磁方法获得的分辨率下被检测到。中间体的寿命必须在毫秒或更长的范围内，才能使用当前的技术，而且确实有许多已知的例子是这样的。 具体目标1是我们继续努力执行和完善现有的时间分辨方式，以便能够有效和富有成效地开展校内和校外合作项目。具体目标2代表了一个全新的创新项目，将与附近伦斯勒理工学院集成电子中心(CIE)的陆东明博士及其同事密切合作，达成合同协议。

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. ABSTRACT The goal of time-resolved cryo-electron microscopy studies is to characterize the structures of intermediates that arise during the functioning of macromolecules. Developments in instrumentation, specimen preparation, and data analysis are carried out using specimens from existing in-house projects that benefit from time-resolved imaging: intermediate states of translating ribosomes, functional states (open, closed, inactivated/adapted) of the ryanodine receptor class of calcium release channel, and metabolic states of the mitochondrion. For the large complex assemblies of macromolecules that are of interest here, some of these intermediates are expected to involve movements of entire domains or subunits, and loss or gain of component subunits (e.g. as in the assembly or disassembly of complexes) or macromolecular ligands (e.g., tRNAs interfacing with the ribosome). These types of structural changes should be detectable at resolutions that are typically attained by the single-particle cryo-EM approach. The lifetime of the intermediates must be in the millisecond range or greater to be accessible with current technology, and there are indeed many examples where this is known to be the case. Specific aim 1 is a continuation of our efforts to implement and refine existing time-resolved modalities so that intramural and extramural collaborative projects can commence efficiently and productively. Specific aim 2 represents an entirely new innovative project that will be done in close collaboration involving a contractual agreement with Dr. Toh-Ming Lu and his associates of the Center for Integrated Electronics (CIE) at the nearby Rensselaer Polytechnic Institute.