Gram-positive (G+) bacteria, such as the Enterococcus species faecium and faecalis, cause around half of hospital-acquired infections. These infections are increasingly antimicrobial-resistant (AMR), which presents a major challenge for tackling the global burden of disease. AMR arises from mutant genes, which can be inherited, and also spread between bacterial species by conjugation through type 4 secretion systems (T4SS), which are protein complex. T4SS are an attractive target for new antibioitics, but development has proven difficult without detailed structural information on these molecular machines. Information is especially lacking for G+ species, which we will address. This project will use cryogenic electron microscopy and tomography to study the T4SS of E. faecalis and obtain the first high-resolution and in situ structures of any G+ T4SS, combining my experience with the cryo-EM of membrane protein complexes and the host’s expertise in G+ T4SS. We will reveal the molecular architecture of a critical conjugation machine and its network of protein-protein interactions. This will enable the precise mechanism of gene transfer and essential (targetable) proteins to be determined.