Nature harnesses fractal geometry to create structures with unusual surface-to-volume ratios. Now, a new design approach enables the reversible assembly of functional enzymes into arboreal patterns with fractal geometry.

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

ABSTRACT

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides’ positions, a task that has previously been accomplished by X-ray crystallography. Here we present a cryo-electron microscopy (cryo-EM) structure of Escherichia coli ( E. coli ) 50S subunit at an average resolution of 2.2Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg 2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for identification of post-transcriptional rRNA modifications using a cryo-EM map. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

This chapter discusses the use of diffraction simulators to improve experimental outcomes in macromolecular crystallography, in particular for future experiments aimed at diffuse scattering. Consequential decisions for upcoming data collection include the selection of either a synchrotron or free electron laser X-ray source, rotation geometry or serial crystallography, and fiber-coupled area detector technology vs. pixel-array detectors. The hope is that simulators will provide insights to make these choices with greater confidence. Simulation software, especially those packages focused on physics-based calculation of the diffraction, can help to predict the location, size, shape, and profile of Bragg spots and diffuse patterns in terms of an underlying physical model, including assumptions about the crystal's mosaic structure, and therefore can point to potential issues with data analysis in the early planning stages. Also, once the data are collected, simulation may offer a pathway to improve the measurement of diffraction, especially with weak data, and might help to treat problematic cases such as overlapping patterns.

In celebration of Pride Month, we asked transgender, genderqueer, and nonbinary scientists to tell us about what fascinates them, their ambitions and achievements, and how their gender identities have shaped their experiences in STEM. We owe a special thanks to 500 Queer Scientists (https://500queerscientists.com/), whose network and efforts at increasing LGBTQ+ scientists' visibility made this article possible.

ABSTRACT

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.