High-resolution cryo-EM structures of plant cytochrome b6fb_{6}f at work

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome $b_{6}f$ (Cyt $b_{6}f$) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site ($Q_{p}$) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt $b_{6}f$ homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near $Q_{p}$ in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt $b_{6}f$ in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration. Structures of cytochrome $b_{6}f$ with and without plastocyanin imply a one-way traffic of quinones for efficient photosynthesis

Molecular basis of tRNA recognition by the Elongator complex

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo–electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer

Cryo-EM structures of the human Elongator complex at work

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action

Cryo-EM structure of the fully assembled elongator complex

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes