Breaking Barriers in Structural Biology: Novel CryoEM Methods and Applications

 描述(申请人提供)：近年来，随着某些以前极具挑战性的生物结构使用该技术变得更加容易处理，单粒子冷冻电子显微镜(CryoEM)的活性和兴趣出现了爆炸性增长。一些结构，如二十面体病毒和核糖体，现在正在常规基础上解析到接近原子的分辨率。这些能力意味着，原子级的结构信息对于许多长期追求的蛋白质靶标来说是潜在的，从而为结构生物学中令人兴奋的发现打开了大门。然而，从本质上讲 采集数据的低信噪比使得使用该技术研究某些目标具有极大的挑战性，并且分辨率充其量将被限制在大范围内。在这一应用中，单粒子低温EM的主要挑战之一将通过开发一种方法来解决，该方法将使小(&lt；100 kDa)大分子和大分子化合物的常规结构解决方案成为可能。同时，现有的技术基础设施，加上方法上的改进，将应用于生物学中的一个突出问题--人类IKK复合体的冷冻EM结构，它是基于核因子-κB的转录调控的中心调节因子，也是药物设计的关键靶点。尽管之前使用基于X射线的技术进行了努力，但IKK的结构以及其激活的合理结构模型仍然难以捉摸。利用低温电子显微镜解决IKK的结构将绕过与样品结晶有关的困难，同时建立在单粒子技术的固有优势上，特别是在它们表征动态和 多相大分子集合体。这项工作将为今后与实验室附近的研究小组合作进行的功能分析奠定基础，并预计将对针对IKK复合体的药物设计工作产生广泛影响。

 DESCRIPTION (provided by applicant): Single-particle cryo-electron microscopy (cryoEM) has witnessed an explosion of activity and interest in recent years, as certain biological structures that were previously extremely challenging to solve have become much more tractable using the technology. Some structures, like icosahedral viruses and ribosomes, are now being solved to near-atomic resolution on a routine basis. The capabilities imply that atomic-level structural information is potentially achievable for many long sought-after protein targets, thus opening doors for exciting discoveries in structural biology. However, the inherently low signal-to-noise ratio of the acquired data makes certain targets extremely challenging to study using the technique, and the resolution will be limited to large domains, at best. In this application, one of the major challenges in single-particle cryoEM will be addressed with the development of a methodology that would enable routine structure solution of small (<100 kDa) macromolecules and macromolecular complexes. In parallel, the existing technological infrastructure, together with methodological improvements, will be applied to an outstanding problem in biology - the cryoEM structure of the human IKK complex, a central regulator of NF-κB based transcription regulation and a key target for drug design. Despite previous efforts using X-ray based techniques, the structure of IKK, and a rational structure-based model of its activation, remains elusive. The utilization of cryoEM to solve the structure of IKK will bypass the difficulties associated with specimen crystallization, while building on the inherent advantages of single-particle techniques, specifically in their ability to characterize dynamic and heterogeneous macromolecular assemblies. This work will provide groundwork for future functional analyses that will be performed in collaboration with research groups in the immediate vicinity of the laboratory and is expected to a broad impact on drug design efforts aimed at the IKK complex.