Breaking barriers in CryoEM through computational protein design

摘要 使用单粒子低温电子显微镜（cryoEM），我们可以研究蛋白质和蛋白质复合物， 原子分辨率和阐明动力学和不同的功能状态在本地或近本地环境。 但这对于小蛋白质来说几乎是不可能的。我的实验室对了解蛋白质功能很感兴趣， 观察它们在自然状态下的高分辨率结构。我们利用冷冻EM和计算方面的专业知识 蛋白质设计，研究蛋白质，可溶性和膜，以及它们的复合物，在人类 疾病膜蛋白是细胞的“守门人”，也是细胞膜蛋白中最重要的一类 蛋白质是G蛋白偶联受体（GPCR），其通过配体诱导的信号传导途径起作用。 细胞外的结合到细胞内结合复合物的募集以传递信号。因为他们参与了 在大多数细胞过程中，它们在药物开发中具有相当大的意义。 虽然cryoEM在结构生物学中获得了动力，但它有一个基本的限制，即蛋白质 小于40 kDa的细胞不能被有效地研究，因为图像中的信号太低。这意味着 人类基因组中的大多数蛋白质不能用冷冻电镜研究。使用设计的方法，我最近 能够通过cryoEM解决17kDa蛋白质的高分辨率结构，几乎比目前的小3倍 cryoEM尺寸限制。我成功了，因为我们使用计算机设计将17kDa的蛋白质连接到支架上 通过增加粒子的质量和更高的对称性， 重建这个原理证明实验证明了使用计算 cryoEM的设计虽然令人兴奋，但这种支架方法仍处于起步阶段， 才能充分发挥其潜力新的支架能够展示重要的膜蛋白， 将制定、测试和优化全球项目完成报告。这些新的纳米材料将专门针对解决 cryoEM需要。通过这种方法，我们将研究膜蛋白结构，描述功能动力学 并促进快速结构导向药物设计，以帮助对抗破坏性 疾病

ABSTRACT Using single-particle cryogenic electron microscopy (cryoEM) we can study proteins and protein complexes to atomic resolution and elucidate dynamics and distinct functional states in native or near-native environments. But this is virtually impossible to do with small proteins. My lab is interested in understanding protein function by observing their structures at high-resolution in native states. We leverage expertise in cryoEM and computational protein design to study proteins, both soluble and membrane, and their complexes that play major roles in human diseases. Membrane proteins are cellular gatekeepers and one of the most important class of membrane proteins are the G protein-coupled receptors (GPCRs), which act as signal conduits through ligand-induced binding on outside cells to the recruitment of binding complexes inside cells to relay signals. As they are involved in most cellular processes they are of considerable interest in drug development. While cryoEM has gained momentum in structural biology, it has a fundamental limitation that proteins smaller than 40 kDa cannot be studied effectively because the signal in the images is too low. This means that most proteins in the human genome cannot be studied by cryoEM. Using a designed approach, I was recently able to solve the high-resolution structure of a 17kDa protein by cryoEM, almost 3 times smaller than current cryoEM size limits. I succeeded because we used computational design to attach the 17 kDa protein to a scaffold which helped imaging by increasing the mass of the particle and the higher symmetry afforded better reconstruction. This proof-of-principal experiment demonstrates the powerful combination of using computational design for cryoEM. While exciting, this scaffold approach is still at its infancy and further design and development are needed to realize its full potential. New scaffolds capable of displaying important membrane proteins like GPCRs will be developed, tested and optimized. These new nanomaterials will be specifically tailored to address cryoEM needs. With this approach we will investigate membrane protein structure, describe functional dynamics in near native environments and facilitate rapid structure-guided drug design to help against devastating diseases.