Department of Structural Biology, Weizmann Inst., Rehovot, Israel
and
Max-Planck-Laboratory for Ribosomal Structure, Hamburg, Germany
The universal cell organelles facilitating the process protein biosynthesis are nucleoprotein assemblies, the ribosomes. A typical bacterial ribosome weighs over 2.3 million daltons and contains up to 73 different proteins and 3 RNA chains of about 4500 nucleotides, arranged in two subunits of unequal size. For illuminating the detailed mechanism of the translation of the genetic code into polypeptide chains, we have initiated crystallographic studies. Diffracting crystals have been grown from ribosomes and their complexes with non-ribosomal components participating in protein biosynthesis, as well as from native, chemically modified and mutated ribosomal subunits, all from thermophilic or halophilic sources, diffracting best to 2.9 Å
X-ray data are being collected with bright synchrotron radiation at cryogenic temperatures from flash-frozen crystals. For phasing by isomorphous replacement methods, heavy atom derivatization is being performed either by soaking crystals in solution of heteropolyanions and multi-metal coordination compounds, or by specific and covalent attachment of monofunctional reagents of dense organo-metalo clusters prior to the crystallization. For the latter approach, as well as at assisting the interpretation of the electron density map, procedures for specific derivatization with rather small and compact clusters are being developed. As there are no exposed cysteines suitable for cluster binding on the surface of the halophilic ribosomes, these are being inserted by site directed mutagenesis. For this aim the surface of these particles are being mapped, procedures for selective and quantitative detachment and reconstitution of several ribosomal proteins. The corresponding genes are being isolated, sequenced and mutated in positions determined by surface-mapping experiments.
Two internal complexes, one composed of a protein and an rRNA fragment and the second of two proteins, have been isolated from the large ribosomal subunit of Haloarcula marismortui. These are being produced for crystallographic analysis.