IMPROVEMENT OF TIME-RESOLVED INSTRUMENTATION

这个子项目是许多利用 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， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要 时间分辨冷冻电子显微镜研究的目标是表征大分子功能过程中出现的中间体的结构。 在仪器，标本制备和数据分析的发展进行使用标本从现有的内部项目，受益于时间分辨成像：翻译核糖体的中间状态，功能状态（开放，关闭，失活/适应）的钙释放通道的兰尼碱受体类，和代谢状态的钙离子。 对于本文感兴趣的大分子的大复杂组装体，预期这些中间体中的一些涉及整个结构域或亚基的移动，以及组分亚基（例如，如在复合物的组装或分解中）或大分子配体（例如，与核糖体相互作用的tRNA）。这些类型的结构变化应该是可检测的分辨率，通常通过单粒子冷冻EM方法获得。 中间体的寿命必须在毫秒范围内或更长，才能用现有技术获得，确实有许多已知情况的例子。 具体目标1是继续努力实施和完善现有的时间分辨模式，使校内和校外的合作项目可以有效和富有成效地开始。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Toh-Ming Lu博士及其同事密切合作。

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.