Bacterial type IV secretion (T4S) systems translocate virulence factors into eukaryotic cells 1,2 , distribute genetic material between bacteria, and have shown potential as a tool for the genetic modification of human cells 3 . Given the complex choreography of the substrate through the secretion apparatus 4 , the molecular mechanism of the T4S system has proven difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy (EM) to reconstruct the T4S system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form a ~3 megadalton (MDa) nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex 1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of twelve VirB4 ATPase subunits organised as side by side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems 1,4-6 .The canonical T4S system comprises 12 proteins, VirB1-11 and VirD4, and forms a large macromolecular complex that spans the cell envelope of Gram-negative bacteria 2 . The hub protein VirB10 inserts into both the inner and outer membranes and spans the entire width of the periplasm. It is decorated by VirB7 and VirB9 in a 1:1:1 ratio to form a C14 symmetrised outer membrane pore termed the core complex 7 . The architecture and relative † Correspondence and requests for materials should be addressed to GW

Type IV secretion systems (T4SSs) are large protein complexes which traverse the cell envelope of many bacteria. They contain a channel through which proteins or protein–DNA complexes can be translocated. This translocation is driven by a number of cytoplasmic ATPases which might energize large conformational changes in the translocation complex. The family of T4SSs is very versatile, shown by the great variety of functions among family members. Some T4SSs are used by pathogenic Gram-negative bacteria to translocate a wide variety of virulence factors into the host cell. Other T4SSs are utilized to mediate horizontal gene transfer, an event that greatly facilitates the adaptation to environmental changes and is the basis for the spread of antibiotic resistance among bacteria. Here we review the recent advances in the characterization of the architecture and mechanism of substrate transfer in a few representative T4SSs with a particular focus on their diversity of structure and function.

DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.

Host infection by pathogenic Mycobacteria such as Mycobacterium tuberculosis is facilitated by virulence factors secreted by Type VII secretion systems. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/Type VII secretion system from Mycobacterium smegmatis at 3.7 Å resolution, resolving the molecular architecture of a Type VII secretion machine and providing insights into the underlying secretion mechanism. The core of the ESX-3 secretion machine consists of four protein components, EccB3:EccC3:EccD3:EccE3 in a 1:1:2:1 stoichiometry, building two identical protomers. The EccC3 coupling protein, which interacts with the secreted substrates, links a flexible array of four ATPase domains to the membrane through a stalk domain. The "domain of unknown function" (DUF) adjacent to the stalk is identified as an ATPase domain essential for secretion. EccB3 is predominantly periplasmatic but a small segment crosses the membrane and contacts the stalk domain, suggesting that conformational changes triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of Type VII secretion systems differs markedly from other known secretion machines.