Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallography

Background: Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscop y (cryo-EM) has yielded fairly detailed three-dimensional reconstructions o f ribosomes. These were used to assist in the determination of higher resol ution structures by X-ray crystallography, Results: Molecular replacement studies using cryo-EM reconstructions provid ed feasible packing schemes for crystals of ribosomes and their two subunit s from Thermus thermophilus, and of the large subunits from Haloarcula mari smortui, For the large subunits,these studies also confirmed the major heav y-atom sites obtained by single isomorphous replacement combined with anoma lous diffraction (SIRAS) and by multiple isomorphous replacement combined w ith anomalous diffraction (MIRAS) at similar to 10 Angstrom. Although adequ ate starting phases could not be obtained for the small subunits, the cryst als of which diffract to 3.0 Angstrom, cryo-EM reconstructions were indispe nsable for analyzing their 7.2 Angstrom multiple isomorphous replacement (M IR) map. This work indicated that the conformation of the crystallized smal l subunits resembles that seen within the 70S ribosomes. Subsequently, crys tals of particles trapped in their functionally active state were grown. Conclusions: Single-particle cryo-EM can contribute to the progress of crys tallography of non-symmetrical, large and flexible macromolecular assemblie s. Besides confirming heavy-atom sites, obtained from flat or overcrowded d ifference Patterson maps, the cryo-EM reconstructions assisted in elucidati ng packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level o f inherent non-isomorphism.