Bactericidal effect of tetracycline in E. coli strain ED1a may be associated with ribosome dysfunction.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.

High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging.

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.

The palisade layer of the poxvirus core is composed of flexible A10 trimers.

Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV.