Tool Development and Applications

摘要 在这个项目中，我们将开发用于确定生物分子三维结构的变革性工具，成像 细胞组装，并将货物运送到目标细胞。纳米技术和结构生物学，特别是 冷冻-EM正在快速发展，我们的目标是为这些革命做出贡献。具体来说，我们将：1) 开发大分子平台，使即使是小蛋白质的结构也能通过 冷冻-EM单粒子方法，平台将是大型、刚性物体，如微管、核糖体或 抗体复合体将与小目标蛋白刚性结合，并增加精确比对所需的质量 粒子，在某些情况下(如微管)，引入高度对称性。2)落实和发展微观经济 用电子衍射(MicroED)1-4和外消旋结晶学方法测定挑战性化合物的结构 目标，5MicroED可以应用于微米大小的晶体，对于标准的X射线结晶学来说太小了。 外消旋结晶学为抵抗初始结晶的小蛋白质(&lt；300残基)提供了希望 屏幕。3)开发可视化病毒及其相关复合体的Clem方法 在细胞内，4)使用被包裹的蛋白纳米囊6来探索病毒萌发和 开发将特定货物运送到细胞中的系统。

ABSTRACT In this Project we will develop transformative tools for determining 3D structures of biomolecules, imaging cellular assemblies, and delivering cargos into target cells. Nanotechnology and structural biology, particularly cryo-EM, are advancing very rapidly, and we aim to contribute to these revolutions. Specifically, we will: 1) Develop macromolecular platforms that will enable the structures of even small proteins to be determined by cryo-EM single particle methods, The platforms will be large, rigid objects like microtubules, ribosomes, or antibody complexes that will bind the small target protein rigidly and add the mass needed to precisely align particles and in some case (like microtubules), introduce high symmetry. 2) Implement and develop micro- electron diffraction (microED)1-4 and racemic crystallographic methods for determining structures of challenging targets,5 MicroED can be applied to micron-sized crystals that are too small for standard X-ray crystallography. Racemic crystallography has promise for small proteins (<300 residues) that resist initial crystallization screens. 3) Develop CLEM methods for visualizing viruses and virus-associated complexes in their native state within cells, 4) Use enveloped protein nanocages6 to explore the fundamental biology of virus budding and develop systems for delivering specific cargoes into cells.