The Saccharomyces cerevisiae mitoribosome synthesizes eight mitochondrial DNA-encoded proteins essential for oxidative phosphorylation. Mitoribosome large subunit (mtLSU) biogenesis involves the conserved DEAD-box helicase Mrh4 and the GTPases Mtg1/GTPBP7 and Mtg2/GTPBP5. Here, we have employed genetic, biochemical, in vitro reconstitution, and cryo-EM approaches to elucidate their hierarchical action during the late stages of mtLSU assembly. We show that Mrh4-mediated bL33m incorporation precedes Mtg1 recruitment to the 21S rRNA. Cryo-EM structures of mitoribosome assembly intermediates accumulating in the absence of Mtg1 or uL16m reveal that Mtg1 restructures the 21S rRNA H73-75 and H93 domains to their mature fold. This subsequently allows the structuring of neighboring peptidyl transfer center region helices and the incorporation of uL6m, uL16m, bL35m, and bL36m during late mtLSU maturation. Unexpectedly, monosomes containing immature mtLSU assemble in Mrh4-, bL33m-, uL16m-, Mtg1-, and Mtg2-depleted mitochondria, at levels that increase with the maturation state of the mtLSU particle. Our data have shed light on the rRNA folding events and the structuring of the MRPs that occur during the late stages of assembly. They have provided insight into the roles of assembly factors Mrh4, Mtg1, and Mtg2 during the process and revealed evolutionarily conserved mechanisms underlying mitochondrial ribosome assembly.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-β-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-β-1,4-mannosidase [EC 3.2.1.25] that is highly specific for β-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Å resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family β-galactosidases and the GH164-family exo-β-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 β-galactosidase-type homotrimeric structure.

Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondria! respiratory supercomplexes III2IV and III2IV2, at 3.2-angstrom and 3.5-angstrom resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Take together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a closed domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.