INITIAL PHASING IN RIBOSOMAL CRYSTALLOGRAPHY. Z. Berkovitch-Yellin1, I. Agmon1, K. Anagnostopoulos2,3, H. Bartels2, A. Bashan1, W.S. Bennett2, vitch-Yellin1, A.Dribin1, F. Franceschi3, H.A.S. Hansen2, J. Harms2, S. Krumbholz2, I. Levin1, S. Morlang3, M. Peretz1, I. Sagi1,3, F. Schlunzen2,R. Sharon1, J. Thygesen2, A. Tocilj1, N. Volkmann2, O. Weinberg1, S. Weinstein1 & A. Yonath1,2. 1Struc. Biol., Weizmann Inst., Rehovot, Israel, 2MPG Lab. for Ribosomal Struc., Hamburg, Germany, and Max-Planck-Inst. for Molecular Genetics, Berlin, Germany3
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 57-73 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 nonribosomal components participating in protein biosynthesis, as well as from native, chemically modified and mutated ribosomal subunits. Those that diffract to the highest resolution obtained so far for ribosomal crystals, 2.9 Å, are of the large ribosomal subunits from Haloarcula marismortui. 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. The suitability of both approaches for phasing at intermediate resolution has been clearly demonstrated in the construction of the following: a 9 Å and 20 Å SIR electron density maps of tbe large ribosomal subunits (of halophilic and meso-thermophilic bacteria, respectively); and a 16 Å MIR map of the small ribosomal subunits from, thermophilic bacteria. The resulting maps show features of size similar to that expected for this particle, and could be further refined by solvent flattening.
Aiming at phasing at higher resolution 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. The corresponding genes are being isolated, sequenced and mutated in positions determined by surface-mapping experiments. In addition, two internal complexes, composed of proteins and rRNA fragments have been isolated. These are being produced for crystallographic analysis.