Micro Electron Diffraction of Toxic and/or Infectious Macromolecular Nanoassemblies

项目摘要 尽管现代结构生物学取得了进步，但许多生物医学相关的结构 大分子组装仍然是遥不可及的或缺乏原子分辨率细节。此外该 确定完全新颖的结构的过程仍然是费力的。所需的大晶体 传统的晶体学实验是一个挑战，增长，并确定 通过X射线晶体学从小的或不完美的晶体获得的结构仍然有限。冷冻- 电子显微镜（cryo-EM）方法有望为高通量 大分子结构测定的方法。低温电磁革命带来了 新的高分辨率方法包括微电子衍射（MicroED）。MicroED 利用电子和纳米级三维晶体之间的强相互作用， 利用新兴的cryo-EM仪器与已建立的晶体学知识。 我的团队帮助实现了MicroED的里程碑式发现，并确定了全新的 蛋白质结构的晶体和晶体碎片小于可见光的波长 光，原子分辨率。这些技术进步，加上更大的可用性， 先进的cryoEM仪器，为进一步改善高- 吞吐量结构确定。制定新的和更有效的办法， 显微电子衍射的结构测定可以为全面探索 复杂的大分子结构仍然是标准方法无法达到的。这些 系统包括生长小的、易碎的或不完美的晶体的大分子复合物。的 与这些类型的组件相关的生物医学问题是多种多样的 通过对疾病的基本解释和作为治疗手段， 平台具体来说，我们的目标是针对感染性和/或有毒的丝状纳米组装体 与淀粉样疾病相关。通过我们的努力，在确定这些具有挑战性的 结构，我们发现启示，以指导改进和发展冷冻电镜 技术，尤其是微电子技术。有了这个目标，我的团队将承担开发 新的高通量方法，用于晶体结构测定，同时获得 以真正的原子分辨率压制生物医学目标的结构。

PROJECT SUMMARY Despite modern advances in structural biology, structures of many biomedically relevant macromolecular assemblies remain out of reach or lack atomic resolution detail. In addition, the process of determining entirely novel structures remains laborious. Large crystals required for conventional crystallography experiments are a challenge to grow, and determination of structures from small or imperfect crystals by x-ray crystallography remains limited. Cryo- electron microscopy (cryo-EM) methods promise to bring new life to high-throughput approaches in macromolecular structure determination. The cryo-EM revolution has brought with it new high-resolution methods including micro electron diffraction (MicroED). MicroED exploits the strong interaction between electrons and nano-scale three-dimensional crystals by leveraging emerging cryo-EM instrumentation against established crystallographic knowledge. My group has helped achieve milestone discoveries in MicroED and determined entirely new protein structures from crystals and crystal fragments smaller than the wavelength of visible light, at atomic resolution. These technological advances, coupled with the greater availability of advanced cryoEM instruments, present an opportunity for further improvement of high- throughput structure determination. The development new and more efficient approaches to structure determination by MicroED could open new avenues for comprehensive exploration of complex macromolecular structures that remain out of reach for standard methods. These systems include macromolecular complexes that grow small, fragile, or imperfect crystals. The biomedical problems associated with these types of assemblies are varied and could broadly impact biomedicine, both through the basic interpretation of disease and as therapeutic platforms. Specifically, we aim to target infectious and/or toxic filamentous nanoassemblies associated with amyloid disease. Through our efforts in determining these challenging structures, we find inspiration to guide the improvement and development of cryoEM techniques, particularly MicroED. With this goal in mind, my group will take on the development of new high-throughput methods for crystallographic structure determination while obtaining structures of pressing biomedical targets at truly atomic resolution.