ADVANCES IN PHASING RIBOSOMAL CRYSTALS

Agmon I¹, Auerbach T¹, Avila H², Bartels H³, Bashan A¹, Bennett WS³, Franceschi F², Hansen HAS³, Harms J³,Janell D³, Kölln I³, Krumbholz S³, Levin I¹, Peretz M¹, Piolleti M², Schlünzen F³, Tocilj A², Weinstein S¹ and Yonath A^1,3

¹Department of Structural Biology, Weizmann Institute, Rehovot
²Max-Planck-Inst. for Molecular Genetics, Berlin
³Max-Planck-Research Unit for Ribosomal Structure, Hamburg; ;

The translation of the genetic code into polypeptide chains may consume up to 80% of the cell's energy and constitute about half of its dry weight. This fundamental life process is performed by more than a hundred components, among them are the giant nucleoprotein assemblies called ribosomes; the universal organelles facilitating the sequential polymerization of amino acids according to the blueprint, encoded in the mRNA. Bacterial ribosomes (m.w. 2.3 mD) are built of two independent subunits of unequal size which associate upon the initiation of protein biosynthesis. The large subunit (1.45 mD) catalyzes the formation of the peptide bond and provides the progression path of the nascent proteins. The small subunit (0.85 mD) contains the site for the initiation of the process and for the decoding of the genetic information. About a 1 I3 of the ribosomal mass comprises of some 58-73 different proteins, depending on its source. The rest 2/3 are three chains of RNA, of a total of about 4500 nucleotides.

Crystals were grown from intact ribosomes and their subunits, despite their unfavorable properties (enormous size, lack of internal symmetry, inherent flexibility and a surface composed of highly degradable RNA alongside with proteins which may be loosely held). Far beyond the initial expectations, two crystal types, of the large ribosomal subunits from Haloarcula marismortui (H50S) and of the small subunit from Thermus thermophilus (T30S), diffract to around 3 A. However, the high resolution is not necessarily linked to diffraction of a high quality. On the contrary, the crystal-type diffracting to the highest resolution (H50S), yields the most problematic diffraction data. The bright SR X-ray beam, necessary for the collection of the higher resolution X-ray diffraction data, causes significant decay even at cryo temperature. Nevertheless, owing to the reasonable isomorphism of the T30S crystals, MIR phases were determined at 7.5 A and led to electron density map which contains long continuous chains that can be traced as RNA chains as well as features that may be interpreted as ribosomal proteins. In contrast, the H50S crystals suffer from substantial radiation sensitivity accompanied by a low level of isomorphism, instability of the unit cell dimensions, deformed spot-shape and non-isotropic mosaicity. The 9.5 A MIR electron density map, constructed to gain insight into this unusual system, may indicate the reasons for the problematic nature of these crystals. Molecular replacement studies, exploiting images reconstructed* from electron micrographs of ribosomal particles embedded in vitreous ice, have been performed for the determination of the packing arrangements of the crystal forms which diffract to lower resolution: the large ribosomal subunit and a complex of the whole ribosome with mRNA and tRNA, both from Thermus thermophilus (T50S and T70S, respectively). The reliability of the so obtained tentative packing arrangements, their verification and their contribution for the determination of the particle's volume (a value not readily obtained by electron microscopy) will be discussed.

* By M. van Heel and H. Stark, Imperial College, London