Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Nucleic Acids Res. 2023 Apr 11;51(6):2862-2876. doi: 10.1093/nar/gkad082.
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 a 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 entire 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 to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.
理解生物大分子复合物的组装原理仍然是一个重大挑战，这是由于系统的复杂性以及开发实验方法的困难。核糖体作为一种核糖核蛋白复合物，是研究大分子复合物组装的理想模型系统。在这项工作中，我们报告了一组在接近生理条件下和共转录的体外重构系统中合成过程中积累的大型核糖体亚基中间结构。使用冷冻电镜单颗粒分析和异质分类，解析了涵盖整个组装过程的 13 个前 50S 中间图谱。对一组密度图谱的分割表明，50S 核糖体中间体是基于十四个协同组装块组装的，其中包括迄今为止报道的最小组装核心，该核心由一个 600 核苷酸长的折叠 rRNA 和三个核糖体蛋白组成。协同块按照特定的依赖性组装到组装核心上，揭示了 50S 亚基早期和晚期组装阶段的并行途径。
