Visualizing translation dynamics at atomic detail inside a bacterial cell

Abstract

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Optimizing Cryo-FIB Lamellas for sub-5Å in situ Structural Biology

“…However, most model systems, such as Saccharomyces cerevisiae ( S. cerevisiae ), Chlamydomonas reinhardtii ( C. reinhardtii ), or Caenorhabdidis elegans ( C. elegans ) are too large to be imaged using transmission electron microscopy directly. In such cases, cryo-focused ion-beam (FIB) milling has become a widespread technique 7–10 . However, averages from cryo-FIB samples which reach sub-5 Å resolution are still the exception and confined to high symmetry proteins or virus particles 11,12 .…”

In situ single particle classification reveals distinct 60S maturation intermediates in cells

“…Examples are the characterization of the spatial organization of a biosynthetic process such as ribosome biogenesis, and the assignment of molecular identities in small volumes such as synapses and vesicles. 3D classification of subtomograms can differentiate between structural states ( Himes and Zhang, 2018 ; Xue et al, 2021 ). However, the assignment of states is unreliable for similar structures that can only be distinguished using high-resolution detail, and statistical approaches to quantitatively assess classification results are lacking.…”

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future

“…In 2020, structures of capsid domain (CA) in lentivirus equine infectious anemia virus (EIAV) immature Gag lattices assembled at two different pH conditions were resolved at sub-4 Å resolution via sub-tomogram averaging (PDB IDs 6t61, 6t64, and 6t63 (Dick et al 2020)). In 2021, 3DEM structures of the 70S ribosome in Mycoplasma pneumoniae cells (PDB IDs 7ood and 7p6z (Xue et al 2021)) were determined at 3.4 Å resolution. These PDB structures highlight the rapid growth and maturation of cryo-ET as a mainstream tool for structure determination at near-atomic resolutions and its potential for revealing complex structures involved in dynamic processes within organelles and cells.…”