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Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane). Science , this issue p. eaas9699 , p. eaat4318 ; see also p. 600 The mechanism by which protons find a path through the key enzyme involved in plant energy generation is elucidated. The chloroplast adenosine triphosphate (ATP) synthase uses the electrochemical proton gradient generated by photosynthesis to produce ATP, the energy currency of all cells. Protons conducted through the membrane-embedded F o motor drive ATP synthesis in the F 1 head by rotary catalysis. We determined the high-resolution structure of the complete cF 1 F o complex by cryo–electron microscopy, resolving side chains of all 26 protein subunits, the five nucleotides in the F 1 head, and the proton pathway to and from the rotor ring. The flexible peripheral stalk redistributes differences in torsional energy across three unequal steps in the rotation cycle. Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark.

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.

Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K + /H + symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins. KUP transporters facilitate potassium uptake by the co-transport of protons and are key players in potassium homeostasis. Here authors identify the potassium importer KimA from Bacillus subtilis as a new member of the KUP transporter family and show the cryo-EM structure of KimA in an inward-occluded, trans-inhibited conformation.

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.

Structural information on the respiratory supercomplex III2IV2 from budding yeast and from Mycobacterium smegmatis has become available, with cryo-EM work from four different groups.

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. 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.

The archaellum is the macromolecular machinery that Archaea use for propulsion or surface adhesion, enabling them to proliferate and invade new territories. The molecular composition of the archaellum and of the motor that drives it appears to be entirely distinct from that of the functionally equivalent bacterial flagellum and flagellar motor. Yet, the structure of the archaellum machinery is scarcely known. Using combined modes of electron cryo-microscopy (cryoEM), we have solved the structure of the Pyrococcus furiosus archaellum filament at 4.2 Å resolution and visualise the architecture and organisation of its motor complex in situ. This allows us to build a structural model combining the archaellum and its motor complex, paving the way to a molecular understanding of archaeal swimming motion.

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis , the K + /H + symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K + concentrations, further K + accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K + binding site and therefore traps KimA in an inward-occluded conformation. Second messengers regulate a broad spectrum of processes in bacteria. Here, authors characterize how the second messenger cyclic di-AMP inhibits the potassium transporter KimA using cryo-EM, molecular dynamics simulations and functional assays.

K + homeostasis is crucial for bacterial survival. The bacterial K+ channel KtrAB is regulated by the binding of ADP and ATP to the cytosolic RCK subunits KtrA. While the ligand-induced conformational changes in KtrA are well described, the transmission to the gating regions within KtrB is not understood. Here, we present a cryo-EM structure of the ADP-bound, inactive KtrAB complex from Vibrio alginolyticus , which resolves part of KtrB’s N termini. They are short intrinsically disordered regions (IDRs) located at the interface of KtrA and KtrB. We reveal that these IDRs play a decisive role in ATP-mediated channel opening, while the closed ADP-bound state does not depend on the N-termini. We propose an allosteric mechanism, in which ATP-induced conformational changes within KtrA trigger an interaction of KtrB’s N-terminal IDRs with the membrane, stabilizing the active and conductive state of KtrAB. KtrAB is a major potassium uptake system that has been linked to the pathogenesis of many infectious bacteria. Here the authors show that KtrB from Vibrio alginolyticus contains an intrinsically disordered N-terminus that is key to its regulation.

Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.