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Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit -a . The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF 1 . Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa. Structural and functional analysis of mitochondria from the human parasite Toxoplasma gondii reveals that its ATP synthase assembles into cyclic hexamers, arranged together in a form of pentagonal pyramids required for maintenance of cristae morphology in Apicomplexa.

The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 Å resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids. Every living thing uses the energy-rich molecule called adenosine triphosphate, or ATP, as fuel. It is the universal molecular currency for transferring energy. Cells trade it, mitochondria make it, and the energy extracted from it is used to drive chemical reactions, transport molecules across cell membranes, energize nerve impulses and contract muscles. ATP synthase is the enzyme that makes ATP molecules. It is a multi-part complex that straddles the inner membrane of mitochondria, the energy factories in cells. The enzyme complex interacts with fatty molecules in the mitochondrial inner membrane, creating a curvature that is required to produce ATP more efficiently. The mitochondrial ATP synthase has been studied in many different organisms, including yeast, algae, plants, pigs, cows and humans. These studies show that most of these ATP synthases are similar to each other, but obtaining a high resolution structure has been a challenge. Some single-cell organisms have unusual ATP synthases, which provide clues about how the enzyme evolved in pursuit of the most energy efficient arrangement. One such organism is the photosynthetic Euglena gracilis, which is closely related to the human parasites that cause sleeping sickness and Chagas disease. Now, Mü̈hleip et al. have extracted ATP synthase from E. gracilis and reconstructed its structure using electron cryo-microscopy. The high resolution of this reconstruction allowed for the first time to examine the fatty molecules associated with ATP synthase, called cardiolipins. This is important, because cardiolipins are thought to modulate the rotating motor of the enzyme and affect how the complex sits in the membrane. The analysis revealed that the ATP synthase in E. gracilis has 29 different protein subunits, 13 of which are only found in organisms of the same family. Some of the newly discovered subunits are glued together by fatty molecules and extend into the surrounding mitochondrial membrane. This distinctive structure suggests an adaptation which likely evolved independently in E. gracilis for efficiency. These results represent an important advance in the field, and provide direct evidence for the functional roles of cardiolipin. This information will be used to reconstruct the evolution of this mighty molecule and to further study the roles of cardiolipin in energy conversion. Moreover, the analysis identified similarities between the ATP synthase in E. gracilis and human parasites, which could provide new therapeutic targets in disease-causing parasites.

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane 1 . Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I–II–III 2 –IV 2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization. A supercomplex comprising all four respiratory chain components contributes to the induction of mitochondrial membrane curvature and tubulation of cristae.

Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF 1 . Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit- a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature. Mitochondrial ATP synthases are involved in shaping the mitochondrial cristae. The cryo-EM structure of type III ATP synthase reveals the architecture of the unusual, asymmetrical, U-shaped dimer and offers insights into the interaction with the natural inhibitor IF1 and membrane lipids. The structure of the enzyme tetramer suggests the mechanism of membrane curvature generation.

F₁Fₒ-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

Mitochondrial ribosomes (mitoribosomes) are essential, and their function of synthesising mitochondrial proteins is universal. The core of almost all mitoribosomes is formed from a small number of long and self-folding rRNA molecules. In contrast, the mitoribosome of the apicomplexan parasite Toxoplasma gondii assembles from over 50 extremely short rRNA molecules. Here, we use cryo-EM to discover the features that enable this unusual mitoribosome to perform its function. We reveal that poly-A tails added to rRNA molecules are integrated into the ribosome, and we demonstrate their essentiality for mitoribosome formation and for parasite survival. This is a distinct function for poly-A tails, which are otherwise known primarily as stabilisers of messenger RNAs. Furthermore, while ribosomes typically consist of unique rRNA sequences, here nine sequences are used twice, each copy integrated in a different mitoribosome domain, revealing one of the mechanisms enabling the extreme mitochondrial genome reduction characteristic to Apicomplexa and to a large group of related microbial eukaryotes. Finally, several transcription factor-like proteins are repurposed to compensate for reduced or lost critical ribosomal domains, including members of the ApiAP2 family thus far considered to be DNA-binding transcription factors. Structure of the Toxoplasma mitoribosome made of >50 short rRNA molecules reveals evolutionary solutions to rRNA fragmentation: addition of poly A tails to rRNAs, reused rRNA sequences, and repurposing of proteins with transcription factor domains.

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit- g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit- g together with subunit- e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit- g/e module as a structural component determining ATP synthase oligomeric assemblies. Mitochondrial ATP synthase assemble into oligomers. Here, authors resolve the structure of trypanosomal ATP synthase, showing that its dimerization is essential for function and evolutionary conserved.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F₁ head, in which the catalytic (αβ)₃ assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

Mitochondrial energy conversion depends on an intricately folded membrane structure, generated by oligomerisation of ATP synthase dimers. However, morphology of cristae membranes varies greatly between different organisms. Recent studies have revealed the unique ATP synthase assemblies of the causative agents of toxoplasmosis and sleeping sickness, giving insight into the role of parasite-specific sub-units in complex assembly, mitochondrial function and parasite fitness.