Structure and Mechanism of the SecYEG-SecDFYajC-YidC Holo-translocon

三分之一的细胞蛋白质组通过无处不在的Sec-机制转运进入或穿过膜。相关因子促进蛋白质移位、插入和折叠。细菌核心SecYEG通道与另外四种膜蛋白SecD、SecF、YajC和YidC组装以形成全转位子分泌酶/插入酶超复合物。全易位子的结构和机制的知识是至关重要的，但难以捉摸的，尽管紧张的努力。我们开发了ACEMBL，一种基于合成生物学的组合多蛋白表达工具，用于产生功能性SecYEG-SecDFYajC-YidC全转位子。生化表征揭示了膜蛋白插入和质子动力依赖性分泌的新机制。最近，我们获得了第一个中等分辨率密度的全易位的cryo-EM令人信服地强调了我们的重组复合物的高品质。我们建议在原子分辨率下确定全转位蛋白分泌酶/插入酶的结构和机制。一系列生物化学，生物物理和工程方法相结合，以实现这一目标。原子信息可以通过X射线晶体学获得，并通过实施革命性的方法，通过冷冻电子显微镜，我们将追求两者协同发现全息translocon架构，底物多肽易位和膜插入，并揭示潜在的机制。我们将使用我们最先进的biosuite（BBSRC资助）进行高通量结晶。我们已经制备的特异性抗体和纳米抗体将用作结晶助剂和构象稳定剂。晶体数据将在国家设施Diamond的高亮度X射线光束线处收集，并在布里斯托进行结构解决方案、改进和建模。与此同时，也为了揭示不同的构象状态，将追求高分辨率冷冻EM。化学交联和纳米抗体将被用于绘制亚基位置和稳定特定构象，以决定性地改善目前的中分辨率EM图近原子的解释。该项目将为最先进的实验策略提供独特的培训机会。这些方法的补充执行将提供非常高的培训潜力，并为未来的学生在分子结构测定的领先方法中获得独特的实验室技能。通过可用的数据体和以协同方法进行跨膜复合物的晶体学和冷冻EM培训的机会，成功前景最大化。第一轮将与科林森和沙菲策尔产生全息translocon复合物，建立在既定的协议。可用的中等分辨率冷冻EM重建（Schaffitzel）将能够与晶体学（Berger）并行处理高分辨率冷冻EM。该项目是及时的-我们最近发表了关于holo-translocon生产和表征的论文，并提交了一份包含低分辨率EM的手稿（部分由BBSRC资助）。

A third of the cellular proteome is transported into or across a membrane, through the ubiquitous Sec-machinery. Associated factors facilitate protein translocation, insertion and folding. The bacterial core SecYEG channel assembles with four further membrane proteins, SecD, SecF, YajC and YidC to form the holo-translocon secretase/insertase supercomplex. Knowledge of holo-translocon structure and mechanism is vital, but elusive in spite of intense efforts. We developed ACEMBL, a synthetic biology-based combinatorial multiprotein expression tool to produce functional SecYEG-SecDFYajC-YidC holo-translocon. Biochemical characterisation revealed new mechanisms of membrane protein insertion and proton-motive-force-dependent secretion. Recently, we obtained first medium resolution density for holo-translocon by cryo-EM compellingly underscoring the high quality of our recombinant complex. We propose to determine the structure and mechanism of holo-translocon protein secretase/insertase at atomic resolution. A range of biochemical, biophysical and engineering approaches are combined to enable this. Atomic information can be obtained by X-ray crystallography and, through implementation of revolutionary methods, by cryo-electron microscopy, and we will pursue both synergistically to discover holo-translocon architecture, substrate polypeptide translocation and membrane insertion, and reveal the underlying mechanisms. We will subject holo-translocon to high-throughput crystallization using our state-of-the-art biosuite (BBSRC funded). Specific antibodies and nanobodies we already prepared will be used as crystallization aids and conformational stabilizers. Crystal data will be collected at high-brilliance X-ray beamlines at the national facility Diamond and structure solution, refinement and model building performed at Bristol. In parallel, also to reveal distinct conformational states, high-resolution cryo-EM will be pursued. Chemical cross-linking and nanobodies will be utilized to map subunit locations and to stabilize specific conformations, to decisively improve the current medium resolution EM map for near atomic interpretation.The project will offer unique opportunities for training in state-of-the-art experimental strategies. Complementary execution of these approaches will offer very high training potential and acquisition of a unique breadth of laboratory skills for the prospective student in the leading methods for molecular structure determination. Success prospects are maximized by body of data available and opportunity to train in crystallography and cryo-EM of a transmembrane complex in a synergistic approach. First rotation will be with Collinson and Schaffitzel to produce holo-translocon complexes, building on established protocols. The available medium-resolution cryo-EM reconstruction(Schaffitzel) will enable tackling high-resolution cryo-EM, in parallel to crystallography (Berger). The project is timely - we have recently published papers on holo-translocon production and characterisation, and submitted a manuscript containing low-resolution EM on holo-translocon (partly BBSRC-funded).