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

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).