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Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Ageing is a progressive decline of intrinsic physiological functions. We examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies (Drosophila melanogaster) by electron cryo-tomography and respirometry. We discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage. DOI: http://dx.doi.org/10.7554/eLife.24662.001

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

Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus. DOI: http://dx.doi.org/10.7554/eLife.07380.001, eLife digest Gram-negative bacteria can cause serious diseases in humans, such as cholera and bacterial meningitis. These bacteria are surrounded by two membranes: an inner membrane and an outer membrane. Proteins called secretins are components of several large molecular complexes that are embedded within the outer membrane. Some secretin-containing complexes form pores in the bacterial membranes and allow molecules to pass in or out of the cell. Some secretins also form part of the machinery that allow Gram-negative bacteria to grow fibre-like structures called type IV pili. These pili help bacteria that cause infections to move and stick to host cells, where they can also trigger massive changes in the host cells' architecture. Multiple copies of a secretin protein called PilQ form a channel in the outer membrane of the bacteria that allows a type IV pilus to grow out of the surface of the cell. The pilus can then hook the bacteria onto surfaces and other cells. There is evidence to suggest the type IV pilus machinery is involved in the uptake of DNA from other bacteria, an important but poorly understood process that has contributed to the spread of multi-drug resistance. Now, Gold et al. have used a cutting-edge technique called ‘electron cryo-tomography’ to analyse the three-dimensional structure of the machinery that builds the type IV pili in the membranes of a bacterium called Thermus thermophilus. This analysis revealed that, similar to many other channel complexes, the PilQ channel can be ‘open’ or ‘closed’. When pili are absent, the channel is closed, but the channel opens when pili are present. Further analysis also revealed the structure of an assembled pilus. Next, Gold et al. studied the open state of the type IV pilus in more detail and observed that a region of each of the PilQ proteins moves a considerable distance to make way for the pilus to enter the central pore. These results will pave the way for future studies of type IV pili and other secretin-containing complexes and underpin efforts to investigate new drug targets to combat bacterial infections. DOI: http://dx.doi.org/10.7554/eLife.07380.002

We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F1Fo ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F1Fo ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F1Fo ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae. DOI: http://dx.doi.org/10.7554/eLife.06119.001, eLife digest Cells use a molecule called adenosine triphosphate (or ATP for short) to power many processes that are vital for life. Animals, plants, and fungi primarily make their ATP in a specialised compartment called the mitochondrion, which is found inside their cells. The mitochondrion is often referred to as the powerhouse of cells as it captures and stores the energy that animals gain from eating food in the molecule ATP. Other enzymes in the cell break apart ATP to release the stored energy, which they use to power various cellular processes. The interior architecture of the mitochondrion includes a highly folded inner membrane where electrical energy is transformed into chemical energy. The tight folding of the inner membrane is thought to make this process more efficient. An enzyme named ATP synthase performs the final steps of the energy transformation process by producing ATP (ATP synthase literally means ‘ATP maker’). This enzyme sits in pairs along the edges of the inner membrane folds. This raises the question: does the ATP synthase cause the membrane to fold or does this enzyme just ‘prefer’ these folded edges (which are instead caused by something else inside the mitochondrion)? To investigate this question, Jiko, Davies et al. extracted ATP synthase from the mitochondria of cow hearts and mixed them with modified fat molecules to form a ‘2D membrane crystal’: a membrane containing an ordered pattern of enzymes. An electron microscope was used to generate a three-dimensional volume of the 2D membrane crystal via a process similar to a MRI or CAT scan that one might have in hospital. In the three-dimensional volume of the membrane crystal, Jiko, Davies et al. discovered that instead of being flat as expected, the membrane of the 2D membrane crystal was rippled and that this ripple was caused by the membrane-embedded part of the ATP synthase. The geometry of the ripple exactly matched half of the bend at the edge of the membrane folds in the mitochondrion. Therefore, Jiko, Davies et al. concluded that a pair of ATP synthases, as found in mitochondria, was responsible for defining the tight folds of the inner mitochondrial membrane. DOI: http://dx.doi.org/10.7554/eLife.06119.002

Sodium/proton antiporters maintain intracellular pH and sodium levels. Detailed structures of antiporters with bound substrate ions are essential for understanding how they work. We have resolved the substrate ion in the dimeric, electroneutral sodium/proton antiporter PaNhaP from Pyrococcus abyssi at 3.2 Å, and have determined its structure in two different conformations at pH 8 and pH 4. The ion is coordinated by three acidic sidechains, a water molecule, a serine and a main-chain carbonyl in the unwound stretch of trans-membrane helix 5 at the deepest point of a negatively charged cytoplasmic funnel. A second narrow polar channel may facilitate proton uptake from the cytoplasm. Transport activity of PaNhaP is cooperative at pH 6 but not at pH 5. Cooperativity is due to pH-dependent allosteric coupling of protomers through two histidines at the dimer interface. Combined with comprehensive transport studies, the structures of PaNhaP offer unique new insights into the transport mechanism of sodium/proton antiporters. DOI: http://dx.doi.org/10.7554/eLife.03579.001, eLife digest Although the membrane that surrounds a cell is effective at separating the inside of a cell from the outside environment, certain molecules must enter or leave the cell for it to work correctly. One way this transport can occur is via proteins embedded in the cell membrane, called transporters. Transporters that are found in all organisms include the sodium/proton antiporters, which exchange protons from inside the cell with sodium ions from outside. However, exactly how the antiporter works was unknown. Previous work suggested that the structure and activity of the sodium/proton antiporter changes as the acidity of its environment changes, but the precise details of how this occurs were unclear. Wöhlert et al. have now crystallised a sodium/proton antiporter from a single-celled organism called Pyrococcus abyssi, a species of archaea that has been found living in hydrothermal vents deep in the Pacific Ocean. The structures the protein takes on in different functional states were then deduced from these crystals using a technique called X-ray crystallography. Using heavy thallium ions instead of sodium ions, which are less visible to X-rays, Wöhlert et al. found the site in the antiporter where the transported ion binds as it moves through the membrane. The antiporter has a funnel-shaped cavity that faces inwards (into the cell) in both acidic and alkaline conditions, although a second narrow channel that is open in alkaline conditions is blocked in acidic conditions by small protein rearrangements. Wöhlert et al. suggest that the differences between both structures explain how the antiporter tunes its ability to bind to the ions it transports. Wöhlert et al. further measured the activity of the antiporter and observed that the transport of ions was most rapid under slightly acidic conditions. In more acidic conditions, the sodium ion cannot bind to the antiporter, and in an alkaline environment, the sodium ions bind too strongly to the antiporter; in both cases, the ions cannot be transported. Comparing the findings presented here with separate work that uncovers the structure of the sodium/proton antiporter in a different species of archaea revealed very similar structures. Related transporters are also found in mammals, and defects in these transporters can lead to problems with the heart and kidneys. A better understanding of the sodium/proton antiporter structure could therefore help to develop new treatments for these conditions. DOI: http://dx.doi.org/10.7554/eLife.03579.002

The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes. DOI: http://dx.doi.org/10.7554/eLife.01963.001, eLife digest Many microbes rely on enzymes known as hydrogenases to catalyse the metabolic reactions that generate energy. These enzymes cleave hydrogen molecules to release electrons that go on to participate in further reactions. In order to fully understand how hydrogenases and other enzymes work it is necessary to work out their structure at the atomic level. Last year a technique known as electron cryo-microscopy (cryo-EM) was used to show that Frh—a hydrogenase that is crucial for many different steps in the metabolic process of microbes that produce methane—had a tetrahedral structure. Cryo-EM involves freezing the molecule of interest in a layer of ice to preserve its structure as it is imaged with an electron beam. Unfortunately, the signal-to-noise ratio in each image is low, so researchers must combine many separate images in order to determine the structure of the molecule. The use of a new type of electron detector can improve the performance of an electron cryo-microscope in several ways. Higher frame rates can be used, which makes it possible to correct for movement of the molecule caused by the electron beam. The new electron detectors are also more efficient, so samples can be exposed to lower doses of electrons, reducing damage to the sample. Using the new direct electron detectors, Allegretti et al. were able to work out the structure of Frh in greater detail than before. The results confirm that the previously reported structure is correct. Furthermore, several new structural features were seen for the first time, including a previously unseen ion located between two protein subunits. Allegretti et al. also revealed that the structure of Frh is highly rigid, and so the process by which it catalyses reactions involving its substrate, the coenzyme F420, does not involve changes in its shape. Instead, the reaction rate depends on the rate at which F420 diffuses to the correct position in the enzyme, where the reaction occurs very rapidly. DOI: http://dx.doi.org/10.7554/eLife.01963.002

We examined substrate-induced conformational changes in MjNhaP1, an archaeal electroneutral Na+/H+-antiporter resembling the human antiporter NHE1, by electron crystallography of 2D crystals in a range of physiological pH and Na+ conditions. In the absence of sodium, changes in pH had no major effect. By contrast, changes in Na+ concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ∼10-fold stronger Na+ binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. Projection difference maps indicated helix movements by about 2 Å in the 6-helix bundle region of MjNhaP1 that is thought to contain the ion translocation site. We propose that these movements convert the antiporter from the proton-bound, outward-open state to the Na+-bound, inward-open state. Oscillation between the two states would result in rapid Na+/H+ antiport. DOI: http://dx.doi.org/10.7554/eLife.01412.001, eLife digest Antiporters are proteins that move molecules or charged particles, such as sodium ions and protons, in and out of cells. Antiporters therefore have an important role in controlling the conditions inside a cell, such as the pH (which is a measure of acidity) and sodium content (which is a measure of saltiness). In human cells, defects in sodium/proton antiport result in heart or kidney failure and other serious diseases. Some aspects of sodium/proton antiporters are well understood, such as the levels of saltiness and acidity that trigger the flow of charged particles in and out of bacterial cells, but the details of how sodium ions or protons activate an antiporter are unknown. Now Paulino and Kühlbrandt have used a technique called electron crystallography to study how the structure of the sodium/proton antiporter changes as the acidity or salt conditions sensed by the antiporter vary. When the concentration of sodium ions was increased at acidic conditions (low pH), the structure of the antiporter began to change so as to increase the ion flow. However, no such changes were observed when the concentration of sodium ions was held constant at a low level while the pH was increased. These findings suggest that, contrary to previous thinking, the operation of a sodium/proton antiporter is largely determined by the concentration of sodium ions. A better understanding of the operation of sodium/proton antiporters should help with the design new treatments for faulty antiporters. DOI: http://dx.doi.org/10.7554/eLife.01412.002