Highlights d 3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate d DNA is trapped between two gates that lead into the cohesin ring d The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state d ATP hydrolysis opens the head gate to complete DNA entry

Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.DOI: http://dx.doi.org/10.7554/eLife.21290.001

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

Mitochondria are the power stations of the eukaryotic cell, using the energy released by the oxidation of glucose and other sugars to produce ATP. Electrons are transferred from NADH, produced in the citric acid cycle in the mitochondrial matrix, to oxygen by a series of large protein complexes in the inner mitochondrial membrane, which create a transmembrane electrochemical gradient by pumping protons across the membrane. The flow of protons back into the matrix via a proton channel in the ATP synthase leads to conformational changes in the nucleotide binding pockets and the formation of ATP. The three proton pumping complexes of the electron transfer chain are NADH-ubiquinone oxidoreductase or complex I, ubiquinone-cytochrome c oxidoreductase or complex III, and cytochrome c oxidase or complex IV. Succinate dehydrogenase or complex II does not pump protons, but contributes reduced ubiquinone. The structures of complex II, III and IV were determined by x-ray crystallography several decades ago, but complex I and ATP synthase have only recently started to reveal their secrets by advances in x-ray crystallography and cryo-electron microscopy. The complexes I, III and IV occur to a certain extent as supercomplexes in the membrane, the so-called respirasomes. Several hypotheses exist about their function. Recent cryo-electron microscopy structures show the architecture of the respirasome with near-atomic detail. ATP synthase occurs as dimers in the inner mitochondrial membrane, which by their curvature are responsible for the folding of the membrane into cristae and thus for the huge increase in available surface that makes mitochondria the efficient energy plants of the eukaryotic cell.