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

Results: Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at ~10 Å. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 Å, cryo-EM reconstructions were indispensable for analyzing their 7.2 Å multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown.