Abstract

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA–protein complexes at near-physiological temperatures.

Highlights

  • The bacterial ribosome possesses universally conserved functional centers that are structurally dynamic and undergo local and large-scale conformational rearrangements during protein synthesis (Ogle et al 2001; Petrov et al 2011; Loveland et al 2017)

  • serial femtosecond X-ray crystallography (SFX) workflow for microcrystals of 30S ribosomal subunits

  • Microcrystals of the 30S ribosomal subunit were soaked with 80 μM paromomycin, 200μM mRNA, and 200 μM phenylalanine tRNA anticodon stem–loop, ASLPhe oligonucleotide, resulting in a slurry of 2 × 2 × 4 μm3 size 30S decoding complex microcrystals

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Summary

Introduction

The bacterial ribosome possesses universally conserved functional centers that are structurally dynamic and undergo local and large-scale conformational rearrangements during protein synthesis (Ogle et al 2001; Petrov et al 2011; Loveland et al 2017). Electron microscopy performed at cryogenic temperature has added even more information about the ribosome during several steps of protein synthesis, at increasing resolution to an existing wealth of structural information spanning several species and conformational states (Mitra et al 2005; Fischer et al 2015; Natchiar et al 2017; Razi et al 2017).

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