Technology Development 1: Development of nanocrystal processing protocols for microED

技术开发计划摘要1 - MicroED 在过去的50年里，X射线晶体学一直是获得原子结构的最成功的方法。 生物分子大的多蛋白质复合物的结晶是具有挑战性的，并且即使成功， 得到的晶体通常小、易碎并存在多种挑战。新的发展，如串行 使用自由电子激光（FEL）和电子晶体学或微电子晶体学的飞秒晶体学 衍射（microED）是获得结构的替代方法，使用纳米或低微米 纳米晶体（nanocrystals）使用自由电子激光解决生物大分子的结构需要数十亿的 纳米晶体，大量的材料，往往是不可用的。使用microED解决结构问题， 另一方面，原则上可以仅使用几个纳米晶体，从而克服与以下有关的问题： 样品数量匹兹堡大学的卡莱罗实验室率先发现了这一发现， 使用透射电子显微镜（TEM）进行优化。这使我们能够大大增加 通过直接观察晶格来确定潜在的结晶条件。在该技术中 发展建议，我们打算发展新的方法来确定蛋白质复合物的结构 通过探索纳米结晶空间和优化， 稳定条件的应用microED方法的结构测定。

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.