Breaking Barriers in Structural Biology: Novel CryoEM Methods and Applications

DESCRIPTION（由申请人提供）：近年来，单粒子低温电子显微镜（cryoEM）的活动和兴趣呈爆炸式增长，因为以前极具挑战性的某些生物结构使用该技术变得更加容易处理。一些结构，如二十面体病毒和核糖体，现在已经可以在常规的基础上以接近原子的分辨率来解决。这种能力意味着，原子水平的结构信息有可能获得许多长期追求的蛋白质目标，从而为结构生物学中令人兴奋的发现打开了大门。然而，所获取数据固有的低信噪比使得使用该技术研究某些目标极具挑战性，并且分辨率最多仅限于大域。在这一应用中，单粒子冷冻电镜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（<100 kDa）大分子和大分子复合物的常规结构解决方案成为可能。与此同时，现有的技术基础设施以及方法的改进将应用于生物学中的一个突出问题-人类IKK复合物的低温结构，这是基于NF-κ b的转录调控的中心调节因子，也是药物设计的关键靶点。尽管先前使用基于x射线的技术进行了努力，但IKK的结构及其合理的基于结构的激活模型仍然难以捉摸。利用冷冻电镜来解决IKK的结构将绕过与样品结晶相关的困难，同时建立在单颗粒技术的固有优势上，特别是在它们表征动态和异质大分子组装的能力上。这项工作将为未来与实验室附近的研究小组合作进行的功能分析提供基础，并有望对针对IKK复合物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.