A humidity-controlled system for reprodicable vitrification of macromolecules

项目摘要 自发现第一种病毒以来近120年，我们对病毒如何将基因组传递到 细胞克服生物膜复杂性的能力仍然有限。虽然大量的科学文献 存在于病毒表面蛋白及其与宿主受体和免疫系统的相互作用上， 重点一直致力于研究将整个病毒基因组递送到细胞中。例如，如何 噬菌体通过革兰氏阴性菌的细胞膜排出DNA？或者，在人类中， 疱疹病毒通过核孔复合体（NPC）将~200 kb的基因组传递到细胞核中？ 在四分之一个世纪里，我先是作为一名实习生（1995-2003），从2004年开始担任首席研究员， 研究了核质转运和病毒基因组包装的机制。我的工作 导致近85篇出版物，有助于阐明原子结构和关键的调控， 与核输入和病毒基因组包装有关的因素。在本R35中，我建议将研究联合收割机 这两个看似不同的生物过程，通过关注病毒基因组传递的机制， 转化成活细胞具体来说，我将提出两个生物学问题，寻求比较和对比如何简单 细菌病毒（或噬菌体）将它们的DNA喷射到细菌中， 将复杂的基因组导入真核细胞的细胞核。第一个问题探讨了噬菌体 通过革兰氏阴性菌的细胞被膜排出约45 kb的基因组。长期以来， 压力驱动注射，这个过程使用了一种病毒编码的纳米机器，我们已经开始研究， 我的实验室第二个问题探讨了疱疹病毒如何将其庞大的基因组通过 核孔复合体（NPC）将人体细胞的细胞核内。这是一种信号和能量介导的 一种利用宿主输入蛋白和GTCRan的过程，利用细胞转运机制促进 外源基因组进入细胞核。总的来说，了解病毒如何转移基因 通过生物膜进入细胞和细胞器的信息对于破译分子 毒力机制以及新型治疗方法的发展。共同 这个R35的分母在于我们对生物的结构和运输机制的兴趣。 大分子我们的研究方法结合了蛋白质生物化学和X射线等成熟科学 晶体学与低温电子显微镜（cryo-EM）的力量，以可视化生物大分子， 接近自然的条件。我们相信，这R35 MIRA的资金机制将燃料的创造性和勤奋 追求我们提出的问题的答案，使我们的研究计划，以实现重大 结构生物学的进步。

Project Summary Nearly 120 years since the discovery of the first virus, our understanding of how viruses deliver genomes into cells overcoming the complexity of biological membranes remains limited. While a vast scientific literature exists on viral surface proteins and their interaction with host receptors, and the immune system, little emphasis has been devoted to studying the delivery of entire viral genomes into cells. For instance, how do bacteriophages eject DNA through the cell envelope of Gram-negative bacteria? Or, in humans, how do herpesviruses deliver ~200 kb genome through the Nuclear Pore Complex (NPC) into the cell nucleus? For a quarter of a century, first as a trainee (1995-2003), and since 2004 as a principal investigator, I have investigated the mechanisms of nucleocytoplasmic transport and viral genome packaging. My work has resulted in close to 85 publications that contributed to elucidating the atomic structure and regulation of crucial factors implicated in nuclear import, and viral genome packaging. In this R35, I propose to combine the study of these two seemingly distinct biological processes by focusing on the mechanisms of viral genome delivery into living cells. Specifically, I will ask two biological questions that seek to compare and contrast how simple bacterial viruses (or bacteriophages) eject their DNA into bacteria with how Herpesviruses deliver their complex genomes into the nucleus of eukaryotic cells. The first question explores how bacteriophages eject ~45 kb genomes through the cell envelope of gram-negative bacteria. Long-thought to be a simple pressure-driven injection, this process uses a virus-encoded nanomachine, which we have begun to study in my laboratory. The second question explores how Herpesviruses deliver their large genome through the Nuclear Pore Complex (NPC) of human cells into the cell nucleus. This is a signal- and energy-mediated process that uses host importins and the GTPase Ran, exploiting the cellular transport machinery to promote entry of an exogenous genome into the nucleus. Overall, understanding how viruses transfer genetic information through biological membranes into cells and organelles is vital for deciphering the molecular mechanisms of virulence as well as the development of novel therapeutic approaches. The common denominator of this R35 lies in our interest in the structure and transport mechanisms of biological macromolecules. Our research approach marries established sciences like protein biochemistry and X-ray crystallography with the power of cryo-electron microscopy (cryo-EM) to visualize biological macromolecules in near-native conditions. We believe that this R35 MIRA funding mechanism will fuel the creative and diligent pursuit of answers to the questions we pose, permitting our research program to achieve significant advancements in structural biology.