RIBOSOMAL CRYSTALLOGRAPHY - FROM CRYSTAL-GROWTH TO INITIAL PHASING

Preliminary phases were determined by the application of the isomorpho us replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosoma l subunits from halophilic and thermophilic bacteria. Derivatization w as performed with dense heavy atom clusters, either by soaking or by s pecific covalent binding prior to the crystallization. The resulting i nitial electron density maps contain features comparable in size to th ose expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron mic roscopy in positively stained thin sections of embedded three-dimensio nal crystals, as well as with phase sets obtained by ab-initio computa tions. Aimed at higher resolution phasing, procedures are being develo ped for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by muta genesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophilic ribosomal subunits whi ch yield crystals diffracting to the highest resolution obtained so fa r for ribosomes, 2.9 and 7.3 Angstrom, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosoma l proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cyste ines) and overexpressed. To assist the interpretation of the anticipat ed electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom bin ding and for attempting the models reconstructed from tilt series of c rystalline arrays of ribosomal particles are being employed for initia l phasing. The tentative functional interpretation of these models sti mulated the design and the crystallization of complexes mimicking defi ned functional states, which were found to be of a higher quality than that obtained from crystals of isolated ribosomes. Specific binding o f multi-metal clusters to these complexes is currently underway.