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<p>Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to <i>E. coli</i>, producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days and facilitating the discovery of a minor population of 100S-like disomes. We envision our approach to be widely applicable to related bacterial samples.</p>

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Rapid structural analysis of bacterial ribosomes in situ

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月28日

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  • Barrett M. Powell,
  • Tyler S. Brant,
  • Joseph H. Davis,
  • Shyamal Mosalaganti

摘要

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli, producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days and facilitating the discovery of a minor population of 100S-like disomes. We envision our approach to be widely applicable to related bacterial samples.