Engineered Affimers for cryo-EM studies of unsolved protein structures

低温电子显微镜（cryo-EM）彻底改变了结构生物学，使我们能够看到具有挑战性的大分子组件的原子结构，包括淀粉样蛋白纤维、SARS-CoV-2和细胞中的蛋白质结构。然而，常规使用低温电子显微镜确定小蛋白质的结构仍然存在挑战。像激酶这样的小蛋白质是药物发现的极好靶点。然而，由于它们的体积小，冷冻电镜对研究这类配体蛋白质的结构不是特别有用。在这个项目中，我们将设计非抗体结合蛋白确定子来开发一个新的工具包和方法来解决这些挑战。Affimers是一项诞生于利兹的技术，已经成功地用于可视化蛋白质和调节它们的功能。我们试图将确认剂的应用扩展到低温电镜。该项目特别侧重于开发一种基于确定子的低温电镜方法，普遍适用于重组蛋白。作为模型靶蛋白，我们将使用40 kDa的有丝分裂激酶Aurora-A，其冷冻电镜结构尚未确定。Aurora-A是有丝分裂的重要调节因子，也是癌症药物发现的重要靶点。因此，该项目将有助于解决基础生物学和人类健康方面的重大挑战。

Cryogenic electron microscopy (cryo-EM) has revolutionised structural biology and has enabled us to see atomic structures of challenging macromolecular assemblies, including amyloid fibres, SARS-CoV-2 and protein structures in cells. However, challenges remain to determine structures of small proteins using cryo-EM routinely. Small proteins such as kinases are excellent targets for drug discovery. However, due to their small size, cryo-EM is not particularly useful to study structures of such proteins with ligands. In this project, we will engineer non-antibody-binding proteins Affimers to develop a new toolkit and methodology that solves these challenges. Affimers are a Leeds-born technology and have been successfully used to visualise proteins and regulate their functions. We seek to extend the application of Affimers to cryo-EM. The project particularly focuses on developing an Affimer-based cryo-EM methodology, universally applicable to recombinant proteins. As a model target protein, we will use a 40 kDa mitotic kinase Aurora-A, of which cryo-EM structure has been undetermined. Aurora-A is an essential regulator of mitosis and a compelling target for cancer drug discovery. Thus, the project will contribute to a major challenge in fundamental biology and human health.