Near-Physiological in vitro Assembly of 50S Ribosomes Involves Parallel Pathways

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as an ideal model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the whole assembly process were resolved using cryo-EM single particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported up to now, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following a defined set of dependencies, revealing the parallel assembly pathways at both early and late assembly stages of the 50S subunit.