A MILESTONE IN RIBOSOMAL CRYSTALLOGRAPHY - THE CONSTRUCTION OF PRELIMINARY ELECTRON-DENSITY MAPS AT INTERMEDIATE RESOLUTION

Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the iso morphous replacement method, have been constructed at intermediate res olution. These maps contain features comparable in size with what is e xpected for the corresponding particles, and their packing arrangement s are in accord with the schemes obtained by ab-initio procedures as w ell as with the motifs observed in thin sections of the crystals by el ectron microscopy. To phase higher resolution data, procedures are bei ng developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mut agenesis or by chemical modifications to facilitate duster binding on the surface of the halophilic large and the thermophilic small ribosom al particles, which yield the crystals diffracting to highest resoluti on (2.9 and 7.3 Angstrom (1 Angstrom = 0.1 nm), respectively). For thi s purpose, the surface of these ribosomal particles is being character ized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core par ticles. The genes of these proteins are being cloned, sequenced, mutat ed to introduce reactive side groups, mainly cysteines, and overexpres sed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstruc ted at low resolution from crystalline arrays of ribosomes and their l arge subunits, are being used for initial low-resolution phasing of th e X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined function al states of a higher quality than those obtained for isolated ribosom es. These models also inspired modelling experiments according to resu lts of functional studies, performed elsewhere, focusing on the progre ssion of nascent proteins.