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

SecYEG-SecDFYajC-YidC Holo-translocon 的结构和机制

基本信息

批准号：
1788547
负责人：
金额：
--
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1788547
关键词：
Structure Mechanism SecYEG SecDFYajC YidC

项目摘要

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).

三分之一的细胞蛋白质组通过无处不在的sec机制进入或穿过膜。相关因子促进蛋白质易位、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组装形成全转座子分泌酶/插入酶超复合物。对全易位结构和机制的了解是至关重要的，但尽管付出了巨大的努力，仍然难以捉摸。我们开发了ACEMBL，这是一种基于合成生物学的组合多蛋白表达工具，用于产生功能性的SecYEG-SecDFYajC-YidC全易位。生物化学特性揭示了膜蛋白插入和质子动力依赖性分泌的新机制。最近，我们通过冷冻电镜（cryo-EM）获得了第一个中等分辨率的全转位密度，有力地强调了我们的重组复合物的高质量。我们建议在原子分辨率上确定全转座子蛋白分泌酶/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，使之成为可能。原子信息可以通过x射线晶体学获得，通过实施革命性的方法，通过冷冻电子显微镜，我们将追求两者的协同作用，以发现全易位结构，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生物套件（BBSRC资助）对全转位子进行高通量结晶。我们已经制备的特异性抗体和纳米体将用作助结晶剂和构象稳定剂。晶体数据将在国家设施的高亮度x射线光束线上收集，并在布里斯托尔进行结构解决、改进和模型构建。同时，为了揭示不同的构象状态，高分辨率冷冻电镜将被追求。化学交联和纳米体将用于绘制亚基位置和稳定特定构象，以决定性地改进当前的中分辨率EM图，用于近原子解释。该项目将为培训最先进的实验策略提供独特的机会。这些方法的互补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主要方法方面获得独特的实验室技能广度。成功的前景是最大的数据体和训练的机会在晶体学和低温电镜跨膜复合体的协同方法。第一次轮换将与科林森和沙菲策尔在既定方案的基础上生产全息-易位子复合物。可用的中分辨率低温电镜重建（Schaffitzel）将能够处理高分辨率低温电镜，与晶体学（Berger）并行。这个项目是及时的-我们最近发表了关于全息转座子生产和表征的论文，并提交了一份包含全息转座子低分辨率EM的手稿（部分由bbsrc资助）。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

DOI：
10.7554/elife.60669
发表时间：
2020-11-04
期刊：
eLife
影响因子：
7.7
作者：
Alvira S;Watkins DW;Troman L;Allen WJ;Lorriman JS;Degliesposti G;Cohen EJ;Beeby M;Daum B;Gold VA;Skehel JM;Collinson I
通讯作者：
Collinson I

Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery.