Abstract
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.
Highlights
X-ray crystallography of the ribosome has played a pivotal role in establishing the structural basis for the mechanism of protein synthesis
The early challenges in obtaining crystals diffracting to a resolution sufficient to provide useful information were overcome at the end of the 1990s (Ramakrishnan, 2010; Steitz, 2010; Yonath, 2010)
Crystals were kept at 277 K before being introduced into the Linac Coherent Light Source (LCLS) beam in a thin liquid jet using a gas dynamic virtual nozzle (GDVN; Fig. 1)
Summary
X-ray crystallography of the ribosome has played a pivotal role in establishing the structural basis for the mechanism of protein synthesis. Ribosomes are large (2.5 MDa) macromolecular assemblies with no internal symmetry, and solving their structures required large well ordered crystals, the development of heavy-atom clusters and the use of synchrotron X-ray sources. Ribosome crystals are highly sensitive to synchrotronradiation damage owing to their high solvent content and large unitcell dimensions with a limited number of crystal lattice contacts, which necessitates longer exposure times This problem was solved by collecting data at cryogenic temperatures (Hope et al, 1989). The current standard synchrotron X-ray cryocrystallography approach has the advantage that ribosomes are in an artificially rigidified state owing to lowered thermal fluctuations, aiding in structure determination It has the disadvantage of potentially masking useful information about local conformational dynamics. Ribosome structural studies would greatly benefit from the ability to use smaller microcrystals at temperatures closer to the physiological range
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