Breaking Barriers in Structural Biology: Novel CryoEM Methods and Applications

描述（由申请人提供）：近年来，单粒子冷冻电子显微镜（cryoEM）的活动和兴趣激增，因为以前非常具有挑战性的某些生物结构已经变得更加易于使用该技术。一些结构，如二十面体病毒和核糖体，现在正被解决到近原子分辨率的常规基础上。这些能力意味着，对于许多长期追求的蛋白质靶点，原子级结构信息是可能实现的，从而为结构生物学中令人兴奋的发现打开了大门。然而，所获得的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，并且分辨率充其量仅限于大范围。在本申请中，单颗粒cryoEM的主要挑战之一将通过开发一种方法来解决，该方法将能够实现小分子（<100 kDa）和大分子复合物的常规结构解决方案。与此同时，现有的技术基础设施，以及方法的改进，将应用于一个突出的问题，在生物学-冷冻电镜结构的人IKK复合物，一个中央调节NF-κB为基础的转录调控和药物设计的关键目标。尽管以前的努力，使用X射线为基础的技术，IKK的结构，和一个合理的结构为基础的模型，其激活，仍然难以捉摸。利用cryoEM来解决IKK的结构将绕过与试样结晶相关的困难，同时建立在单颗粒技术的固有优势上，特别是在其表征动态和异质大分子组装体的能力上。这项工作将为未来的功能分析提供基础，这些分析将与实验室附近的研究小组合作进行，预计将对针对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.