CryoEM Core

核心3：低温EM 摘要 自CRNA问世以来，低温电子显微镜一直是一项关键的结构生物学技术 通过CryoEM核心提供。在CRNA 1.0期间，我们在开发CryoEM方面取得了许多进展 研究小RNA结构的程序，启动了几个CRNA的低温EM项目 研究人员，并举办了低温EM研讨会，以激励CRNA成员和其他人使用这种强大的结构 技术。我们最显著的成就是达到了一个重大的技术里程碑 30 kDa HIV-1二聚体RNA二聚体的亚纳米分辨低温电子显微镜结构 修订当前的Rev响应元件(RRE)结构，并采用严格的CryoEM结构验证协议。 这些研究是与多个CRNA组织合作完成的，其中包括Marchant、Summers和 案例，以整合来自冷冻EM，核磁共振和分子动力学的结构信息。DIS结构 代表了迄今为止由CryoEM确定的最小的大分子结构。进入CRNA 2.0，我们 已经将我们的团队扩大到包括来自贝勒学院的具有互补专业知识的调查人员 医学院、弗吉尼亚大学和美国国立卫生研究院。我们设定了三个目标：(I)利用最先进的低温EM作为一种 用于确定RNA、RNA-蛋白质复合体和HIV的可能最高分辨率结构的工具- 相关蛋白质复合体，与CRNA项目的研究人员合作，并在整个 CRNA；(Ii)在低温EM中开发新的实验和计算协议，以表征挑战性 RNA和RNA-蛋白质复合体的样品，它们在组成或构象上都很小 可变，或两者兼而有之；以及(Iii)整合来自核磁共振、X射线结晶学、低温电子显微镜、分子 与CRNA研究相关的部分或完整生物复合体的动力学模拟和生物化学 以了解它们的结构和功能。此外，我们还将继续提供系统的、 对CRNA学生和博士后进行动手冷冻EM培训，使他们变得更加多才多艺 未来几十年的结构生物学家。

Core 3: CryoEM Summary Since the CRNA’s inception, cryo-electron microscopy (cryoEM) has been a key structural biology technology provided through the CryoEM Core. During CRNA 1.0 we made many advances in developing cryoEM procedures for studying the structures of small RNAs, initiated CryoEM projects with several CRNA investigators, and held cryoEM workshops to inspire CRNA members and others to use this powerful structure technique. Our most notable achievements are reaching a major technical milestone in determining a subnanometer resolution cryoEM structure of the 30 kDa HIV-1 dimerization initiation site (DIS) RNA dimer and revising the current Rev response element (RRE) structure with rigorous cryoEM structure validation protocols. These studies were done in collaboration with multiple CRNA groups, including Marchant, Summers and Case, to integrate structural information from cryoEM, NMR and molecular dynamics. The DIS structure represents the smallest macromolecule structure determined to date by cryoEM. Moving into CRNA 2.0, we have extended our team to include investigators with complementary expertise from Baylor College of Medicine, the University of Virginia and NIH. We have set three aims: (i) to utilize state-of-the-art cryoEM as a tool for determining the highest possible resolution structures of RNAs, RNA-protein complexes and HIV- related protein complexes, in collaboration with investigators from the CRNA projects and throughout the CRNA; (ii) to develop new experimental and computational protocols in cryoEM for characterizing challenging specimens of RNA and RNA-protein complexes, which are either small, compositionally or conformationally variable, or both; and (iii) to integrate measurements from NMR, X-ray crystallography, cryoEM, molecular dynamics simulation, and biochemistry for partial or intact biological complexes relevant to the CRNA research missions in order to understand their structure and function. In addition, we will continue to offer systematic, hands-on cryoEM training to CRNA students and postdoctoral fellows so that they will become more versatile structural biologists in the coming decades.