Metal Compounds as Tools for the Construction and the Interpretation of Medium-Resolution Maps of Ribosomal Particles Shulamith Weinstein,* Werner Jahn,† Carola Glotz,‡ Frank Schlu ¨ nzen,§ Inna Levin,* Daniela Janell,§ Jo ¨ rg Harms,§ Ingo Ko ¨ lln,§ Harly A. S. Hansen,§ Marco Glu ¨ hmann,§ William S. Bennett,§ Heike Bartels,* , § Anat Bashan,* Ilana Agmon,* Maggie Kessler,* Marta Pioletti,‡ Horacio Avila,‡ Kostas Anagnostopoulos,‡ Moshe Peretz,* Tamar Auerbach,* , § Francois Franceschi,‡ and Ada Yonath* , § *Department of Structural Biology, Weizmann Institute, 76100 Rehovot, Israel; †Max-Planck-Institute for Medical Research, Jahn Strasse 29, 69120, Heidelberg, Germany; ‡Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany; and §Max-Planck-Research Unit for Ribosomal Structure, Notkestrasse 85, 22603 Hamburg, Germany Received January 25, 1999, and in revised form April 30, 1999 Procedures were developed exploiting organome- tallic clusters and coordination compounds in com- bination with heavy metal salts for derivatization of ribosomal crystals. These enabled the construction of multiple isomorphous replacement (MIR) and multiple isomorphous replacement combined with anomalous scattering medium-resolution electron density maps for the ribosomal particles that yield the crystals diffracting to the highest resolution, 3 Å, of the large subunit from Haloarcula marismor- tui and the small subunit from Thermus thermophi- lus. The first steps in the interpretation of the 7.3-Å MIR map of the small subunit were made with the aid of a tetrairidium cluster that was covalently attached to exposed sulfhydryls on the particle’s surface prior to crystallization. The positions of these sulfhydryls were localized in difference Fou- rier maps that were constructed with the MIR phases.  1999 Academic Press Key Words: ribosomes; crystallography of ribo- somes; tetrairidium cluster; Ta 6 Br 14 . INTRODUCTION Ribosomes are the supramolecular assemblies re- sponsible for one of the most fundamental life pro- cesses, the translation of the genetic code into pro- teins. The ribosomes are giant nucleoprotein organelles built of two independent subunits of unequal size that associate upon the initiation of protein biosynthesis. In bacteria their molecular mass is about 2.3 MDa and they are composed of RNA and proteins at a 2:1 ratio. The large subunit, called 50S 1 in prokaryotes, is 1.45 MDa, contains two RNA chains with a total of 3000 nucleotides, and contains 38–50 different proteins. It catalyzes the formation of the peptide bond and provides the path for the progression of nascent proteins. The small subunit, 30S, is 0.85 MDa and consists of one RNA chain of 1500 nucleotides and 21 proteins. It offers the site for the initiation and the progression of the process of protein biosynthesis and facilitates the decoding of the genetic information. Crystals have been grown from intact prokaryotic ribosomes and their subunits. Diffraction to around 3 Å was obtained from two crystal types: the large ribosomal subunits from Haloarcula marismortui, H50S (von Bo ¨hlen et al., 1991), and the small subunits from Thermus thermophilus, T30S (Yonath et al., 1998; Harms et al., 1999). Experience showed that the complications in the elucidation of the high-resolution structure of the ribosome are linked not only to the huge size of the ribosomes and to the lack of internal symmetry. They stem primarily from the inherent flexibility and high instability of the ribosomes, since their surface is composed of highly degradable RNA alongside proteins that may be loosely held. These undesired properties are accom- 1 Abbreviations used: 70S, 50S, 30S: the whole ribosome and its two subunits from prokaryotes. A letter as a prefix to the ribosomal particles or ribosomal proteins represents the bacterial source (e.g., E, E. coli; T, Thermus thermophilus; H, Haloarcula marismortui). tRNA and rRNA: transfer and ribosomal RNA. The names of the ribosomal proteins are composed of L or S (showing that this protein is of the large or small subunit) and a running number, according to the position of this protein on the two- dimensional gels. SR: synchrotron radiation; MIR: multiple isomor- phous replacement; MIRAS and SIRAS: multiple and single isomorphous replacement combined with anomalous scattering; MAD: multiwavelength anomalous dispersion; MR: molecular replacement. The chemical formulae of the undecagold and the tetrairidium clusters (including their reactive bridging arms), as well as those of TAMM, PIP, WAC, Hg 6 , Hg 3 , W12, W17CsCo, W18, and W30, are given in Table I. Journal of Structural Biology 127, 141–151 (1999) Article ID jsbi.1999.4135, available online at http://www.idealibrary.com on 141 1047-8477/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.