National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM136412
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM053757
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
AI170855
米国
引用
ジャーナル: Nucleic Acids Res / 年: 2023 タイトル: Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways. 著者: Xiyu Dong / Lili K Doerfel / Kai Sheng / Jessica N Rabuck-Gibbons / Anna M Popova / Dmitry Lyumkis / James R Williamson / 要旨: 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 ...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.
詳細: Most of the sucrose was removed by spin concentration.
凍結
凍結剤: ETHANE / チャンバー内湿度: 80 % / チャンバー内温度: 298 K / 装置: GATAN CRYOPLUNGE 3 / 詳細: 3 microliter of the sample was added..
詳細
The in vitro assembled large ribosomal subunit was purified by sucrose gradient and was spin-concentrated in a 100 kDa MW-cutoff filter.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
詳細
In order to account for highly preferred orientation of the specimen, data was acquired using tilts ranging from 10-60 degrees, in addition to untilted images at 0 degrees. The CTF was estimated on a per-particle basis to account for the gradient of CTF values across individual micrographs