Breaking barriers in CryoEM through computational protein design

摘要 利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体 原子分辨率和阐明在自然或近自然环境中的动力学和独特的功能状态。 但对于小蛋白质来说，这几乎是不可能的。我的实验室对了解蛋白质的功能很感兴趣 在自然状态下以高分辨率观察它们的结构。我们利用在冷冻EM和计算方面的专业知识 蛋白质设计，研究在人体内起主要作用的蛋白质，包括可溶性蛋白质和细胞膜蛋白质及其复合体 疾病。膜蛋白是细胞的守门人，也是最重要的一类膜 蛋白质是G蛋白偶联受体(GPCRs)，通过配体诱导作为信号通道 结合细胞外到细胞内的结合复合体的募集来传递信号。因为他们牵涉其中 在大多数细胞过程中，它们对药物开发有相当大的兴趣。 虽然低温电子显微镜在结构生物学方面获得了发展势头，但它有一个根本限制，即蛋白质 小于40 kDa的图像无法有效研究，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低温电子显微镜研究。使用一种设计的方法，我最近 能够用低温电子显微镜解决一个17 kDa蛋白质的高分辨率结构，几乎比目前的电流小3倍 低温EM大小限制。我成功了，因为我们使用计算设计将17 kDa蛋白质固定在支架上 这有助于通过增加粒子的质量来成像，对称性越高，提供的效果就越好 重建。这一证明原则的实验演示了使用计算的强大组合 为低温EM设计。虽然令人兴奋，但这种脚手架方法仍处于起步阶段，还处于进一步的设计和开发阶段 是实现其全部潜力所必需的。新型支架能够展示重要的膜蛋白，如 将开发、测试和优化GPCRs。这些新的纳米材料将专门为解决 低温EM的需求。用这种方法，我们将研究膜蛋白结构，描述功能动力学 在接近本地环境中，并促进快速结构制导药物设计，以帮助防止毁灭性 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.