Ziva Berkovitch-Yellin

Characterization and preliminary attempts for derivatization of crystals of large ribosomal subunits from Haloarcula marismortui diffracting to 3 A resolution.

An improved form of crystals of large (50 S) ribosomal subunits from Haloarcula marismortui, formally named Halobacterium marismortui, diffracting to 3 A resolution, has been obtained by the addition of 1 mM-Cd2+ to the crystallization medium, which contained more than 1.9 M of other salts. The improved crystals, grown from functionally active particles to an average size of 0.3 mm x 0.3 mm x 0.08 mm, are isomorphous with the previously reported ones, which diffracted to 4.5 A. They are of space group C222(1), cell dimensions a = 210 A, b = 300 A, c = 581 A, and contain one particle in the asymmetric unit. Their superior internal order is reflected not only in their high resolution, but also in their reasonable mosaicity (less than 0.3 degrees). In contrast to the previously grown crystals, the new ones are of adequate mechanical strength and survive well the shock-cooling treatment. Due to their weak diffracting power, all crystallographic studies have been performed with synchrotron radiation. At cryotemperature, these crystals showed no measurable decay for a few days of irradiation and a complete diffraction data set could be collected from a single crystal. Efforts for initial phasing by specific and quantitative derivatization with super-dense heavy-atom clusters are in progress.

Large van der Waals ions

Model calculations of the potential surfaces of van der Waals ions, consisting of a benzene cation bound to a rare‐gas (R) atom, provide information concerning the reduction of the ionization potentials of benzene in the benzene–R1 complex, relative to that of the bare molecule, and establish the configuration changes accompanying the ionization of benzene–R1 complexes.

Hollows, voids, gaps and tunnels in the ribosome

Renewed discussion of concepts hitherto regarded as sacred icons has made the past year an exciting period in the study of the translation apparatus. In particular, reconstructed models of the ribosome have revealed key structural features, associated with empty or partially filled hollows, which, in turn, have stimulated functional assignments of varying degree of objectivity.

Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus.

Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus. These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm. Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation. They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules. Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

Aspects in Structural Studies on Ribosomes

(1992). Aspects in Structural Studies on Ribosomes. Critical Reviews in Biochemistry and Molecular Biology: Vol. 27, No. 4-5, pp. 403-444.

Energetics and dynamics of large Van der Waals molecules

We report on the synthesis, identification, excited-state energetics, interstate electronic relaxation and intrastate nuclear dynamics in electronically–vibrationally excited states of Van der Waals molecules, consisting of a large aromatic molecule bound to inert-gas atoms.

The interplay between X-ray crystallography, neutron diffraction, image reconstruction, organo-metallic chemistry and biochemistry in structural studies of ribosomes

Crystals of ribosomes, their complexes with components of protein biosynthesis, their natural, mutated and modified subunits, have been subjected to X-ray and neutron crystallographic analyses. Electron microscopy and 3-dimensional image reconstruction, supported by biochemistry, genetic, functional and organo-metallic studies were employed for facilitating phasing of the crystallographic data. For example, a monofunctional multi heavy-atom cluster (undecagold) was designed for covalent and quantitative binding to ribosomes. The modified particles were crystallized isomorphously with the native ones. Their difference-Patterson maps contain indications for the usefulness of these derivatives for subsequent phasing. Models of the ribosome and its large subunit were reconstructed from tilt series of 2-dimensional sheets. The comparison of the various reconstructed images enabled an initial assessment of the reliability of these models and led to tentative assignments of several functional features. These include the presumed sites for binding mRNA and for codon-anticodon interactions, the path taken by the nascent protein chain and the mode for tRNA binding to ribosomes. These assignments assisted in the design of biologically meaningful crystal systems. The reconstructed models are being used to identify structural features in initial density maps derived from X-ray and neutron diffraction data.

Towards Atomic Resolution of Prokaryotic Ribosomes: Crystallographic, Genetic and Biochemical Studies

The studies reported here were initiated and inspired by the late Prof. H.G. Wittmann. From the early stages of this project, when it was widely believed that even the initial steps in determining the molecular structure of ribosomes are impossible, until his last days, Prof. Wittmann was actively involved in the experimental design and in the actual studies. We have no doubt that without his motivation, optimism, guidance and support, this project would not have reached its current stage.

MORPHOLOGY ENGINEERING OF ORGANIC CRYSTALS WITH THE ASSISTANCE OF “TAILOR-MADE” GROWTH INHIBITORS

Abstract Retardation of growth of organic crystals in the presence of additives in solution has been investigated in terms of their crystal structures and the stereochemistry of the additives. A simple correlation has been established between affected directions of growth and structure of the additive. This mecnanism has been successfully applied for monitoring, in a controlled manner, the morphology of organic crystals; this is illustrated for benzamides and benzoic acids. Furthermore, the absolute configuration of polar crystals and chiral molecules has been directly assigned. This is illustrated for the polar crystal of tr-cinnamoyl alanine, and for the molecule of threonine, through morphological changes this molecule induces in the centrosymmetric crystal of serine.

Derivatization of ribosomes and of tRNA with an undecagold cluster: crystallographic and functional studies

An undecagold cluster was covalently attached to whole ribosomes and to their small and large subunits prior to their crystallization. X-ray crystallographic data were collected from crystals of the first two. The same cluster was bound to tRNAphe fromE. coli at base 47. It was found that the modified tRNA molecule binds to the ribosome and can be aminoacylated by its cognate synthetase. The gold cluster modified tRNAphe may be used for phasing diffraction data of crystals of complexes containing it, mimicking defined states in the process of protein biosynthesis.