Evolution of ribosome X-ray crystallography: from first crystals to atomic resolution

X-ray crystallography has been pivotal in elucidating the structural basis of ribosome function and in informing antibiotic design, from early challenges in purifying and crystallizing these mega-Dalton assemblies to recent advances integrating synchrotron radiation and X-ray free-electron lasers for high-resolution and time-resolved studies. Methodological innovations - including stress-induced lattice packing, extremophile ribosome sources, optimization of the purification procedure and vapor-diffusion screens - have yielded crystals diffracting up to 2.4 Å. Progress in phasing strategies, from multiple isomorphous replacement and multiwavelength anomalous dispersion/single-wavelength anomalous dispersion to cryogenic electron microscopy-guided molecular replacement and serial femtosecond crystallography, has overcome the phase problem for large complexes. Landmark structures of prokaryotic and eukaryotic ribosomes have elucidated the architectural principles underlying peptide bond formation and messenger RNA decoding, leading to inhibitor discovery. Despite the rise of cryogenic electron microscopy, crystallography remains indispensable for mechanistic insights and rational drug design targeting ribosomal sites.