Structural studies of eukaryotic protein synthesis factor complexes by Cryo EM

分子识别推动了几乎所有的生物反应。该项目旨在揭示所有真核细胞中对翻译至关重要的蛋白质因子之间相互作用的结构细节。具体的翻译启动因子是G蛋白eIF2和两个称为eIF2B和eIF5的调节因子。EIF2是启动子tRNA与核糖体结合所必需的。其GTP/GDP地位由eIF2B和eIF5控制。EIF2在特定丝氨酸残基上的磷酸化通过形成非生产性的eIF2：eIF2B复合体来抑制eIF2B的活性。这种翻译控制机制在所有被研究的真核生物中都是保守的，在包括发育、新陈代谢和长期记忆形成在内的不同环境中都是重要的。EIF2和eIF2B都是大的多亚单位复合体，这些因子的突变会导致神经系统疾病。我们的目标是解构这些复合体的作用机制及其调控过程。纯化的翻译因子复合体之间的结构和相互作用将通过最先进的分子冷冻电子显微镜技术来阐明。该项目将尖端的生物物理分析方法与创新的数据分析相结合。它将把现代精密分子生物学技术与生物化学和酵母遗传学结合起来。该项目涉及蛋白质表达和纯化、分子生物学、电子显微镜、3D结构分析和分子模型建立。该项目将需要数据和统计信息的3D可视化，以可靠地解释不同蛋白质复合体中的微小差异。我们的目标是获得有关蛋白质复杂运动和动态信息以及单个结构的信息。将开发新的可视化方法，以及新的定量3D图像分析方法。

Molecular recognition drives almost all biological reactions. This project aims to uncover structural details of the interactions between protein factors critical for translation in all eukaryotic cells. The specific translation initiation factors are the G protein eIF2 and two regulators called eIF2B and eIF5. eIF2 is required to bind initiator tRNA to ribosomes. Its GTP/GDP status is controlled by eIF2B and eIF5. Phosphorylation of eIF2 at a specific serine residue inhibits eIF2B activity by forming a non-productive eIF2:eIF2B complex. This mechanism of translational control is conserved across all studied eukaryotes and is important within diverse settings including developmental, metabolism and long-term memory formation. eIF2 and eIF2B are both large multi-subunit complexes, and mutations within these factors cause neurological diseases. We aim to deconstruct the mechanism of action of these complexes, and their regulation processes. The structure and interactions between purified translation factor complexes will be elucidated by state-of-the-art molecular cryo electron microscopy techniques.This project incorporates cutting-edge biophysical analysis methods with innovative data analysis. It will blend modern precision molecular biology techniques with biochemistry and yeast genetics. The project involves protein expression and purification, molecular biology, electron microscopy, 3D structure analysis and molecular model building.This project will require 3D visualisation of data and statistical information to reliably interpret small differences in different protein complexes. We aim to get information on protein complex motion and dynamic information, as well as individual structures. Novel visualisation methods will be developed, as well as new quantitative 3Dimage analysis methods.