Technology Development 1: Development of nanocrystal processing protocols for microED

技术发展计划摘要1-MicroED 在过去的50年里，X射线结晶学一直是获得原子结构的最成功的方法 生物分子。大型多蛋白质复合体的结晶是具有挑战性的，即使成功， 产生的晶体通常很小，很精致，面临着多重挑战。新的发展，如连载 使用自由电子激光(FEL)的飞秒结晶学和电子结晶学或微电子 衍射(MicroED)是获得结构的替代方法，使用纳米或低微米 大小的晶体(纳米晶体)。用自由电子激光解算生物大分子结构需要数十亿美元 纳米晶体，大量经常无法获得的材料。使用MicroED解决结构问题，在 另一方面，原则上可能只用几个纳米晶体就可以，从而克服了与 样本量。匹兹堡大学的卡莱罗实验室率先发现了纳米晶体，并 用透射电子显微镜进行了优化。这使得大大增加了 通过直接观察晶格来确定潜在结晶条件的数量。在这项技术中 开发计划，我们打算开发新的方法来确定蛋白质复合体的结构 通过探索纳米结晶空间和优化HIV-1蛋白及其人类结合伙伴 微电子能谱方法用于结构测定的稳定条件。

Abstract for Technology Development Program 1 - MicroED X-ray crystallography over the last 50 years has been the most successful method to obtain atomic structures of biological molecules. Crystallization of large multi-protein complexes is challenging, and even if successful, the resulting crystals are usually small, delicate and present multiple challenges. New developments such as serial femtosecond crystallography using a free electron laser (FEL) and electron crystallography or micro-electron diffraction (microED) are alternative approaches to obtain structures, using nano-meter or low micro-meter sized crystals (nanocrystals). Solving structures of bio-macromolecules using FELs requires billions of nanocrystals, amounts of material that frequently is not available. Solving structures with microED, on the other hand, may, in principle, be possible with only a few nanocrystals, thus overcoming problems related to sample quantity. The Calero laboratory at the University of Pittsburgh has pioneered nanocrystal discovery and optimization using transmission electron microscopy (TEM). This has permitted to significantly increase the number of potential crystallization conditions through direct observation of crystal lattices. In this technology development proposal, we intend to develop new methodologies to determine structures of protein complexes of HIV-1 proteins and their human binding partners by exploring nano crystallization space and optimizing stabilizing conditions for the application of microED approaches for structure determination.