Structural studies of ribosome-SelB complexes and ribosome nascent chain folding intermediates by cryo-EM and molecular dynamics simulations

在P2中，我们将重点关注核糖体复合物的高分辨率单颗粒冷冻EM结构测定与随后通过分子动力学模拟进行的详细分析的结合。具体来说，我们将讨论两种不同的核糖体复合物。我们的第一个主题是专注于硒代半胱氨酸掺入的延伸因子SelB和SelB与核糖体相互作用的结构动力学。我们的第二个目标是研究共翻译蛋白折叠的模型蛋白NemK在高分辨率的时间分辨冷冻EM研究和模拟。核糖体的结构现在可以通过cryo-EM以直接允许原子模型建立的分辨率来解决，并且由于电子显微镜硬件和图像分析的最新改进，在延伸因子（SelB）结合期间或在蛋白质折叠期间处于不同功能状态的核糖体的中间结构在该分辨率水平下变得可访问。这是通过分子动力学模拟详细分析动态事件的起点，其提供了关于不同功能状态之间的能垒和速率常数的定量信息，并最终有助于我们理解核糖体功能中的驱动力。因此，cryo-EM和MD模拟的组合是独特的，以了解延伸因子结合和共翻译蛋白质折叠的分子过程，并获得详细的见解，在近原子分辨率的核糖体的动力学。

In P2 we will focus on the combination of high-resolution single particle cryo-EM structure determination of ribosome complexes and the subsequent detailed analysis by molecular dynamics simulations. Specifically we will address two different ribosome complexes. Our first topic is focused on selenocysteine incorporation by the elongation factor SelB and the structural dynamics of SelB interaction with the ribosome. Our second aim is to study co-translational protein folding of the model protein NemK at high resolution by time-resolved cryo-EM studies and simulations. The structure of the ribosome can now be solved by cryo-EM at resolutions that directly allow atomic model building and intermediate structures of ribosomes in different functional states during elongation factor (SelB) binding or during protein folding become accessible at this resolution level due to the latest improvements in electron microscopy hardware and image analysis. This is the starting point for detailed analysis of dynamic events by molecular dynamics simulations which provides quantitative information on energy barriers and rate constants between different functional states and finally contributes to our understanding of driving forces in ribosome function. The combination of cryo-EM and MD simulations is therefore unique to understand the molecular processes of elongation factor binding and co-translational protein folding and to obtain detailed insights into the dynamics of the ribosome at near-atomic resolution.