Shulamith Weinstein

Crystallographic Studies on the Ribosome, a Large Macromolecular Assembly Exhibiting Severe Nonisomorphism, Extreme Beam Sensitivity and No Internal Symmetry

Crystals, diffracting best to around 3 A, have been grown from intact large and small ribosomal subunits. The bright synchrotron radiation necessary for the collection of the higher-resolution X-ray diffraction data introduces significant decay even at cryo temperatures. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small ribosomal subunits, reliable phases have been extracted at medium resolution (5-6 A) and an interpretable five-derivative MIR map has been constructed. For the crystals of the large subunits, however, the situation is more complicated because at higher resolution (2.7-7 A) they suffer from substantial radiation sensitivity, a low level of isomorphism, instability of the longest unit-cell axis and nonisotropic mosaicity. The 8 A MIR map, constructed to gain insight into this unusual system, may provide feasible reasoning for the odd combination of the properties of these crystals as well as hints for future improvement. Parallel efforts, in which electron-microscopy-reconstructed images are being exploited for molecular-replacement studies, are also discussed.

N-acyl derivatives of chiral amines as novel, readily prepared phases for the separation of optical isomers by gas chromatography

Abstract The results reported demonstrate that it suffices for a chiral stationary phase to contain an amide group and an asymmetric carbon atom, attached to the nitrogen atom [RCONHCH(CH3)R′], in order to show selectivity in its interaction with the enantiomers of amides such as N-trifluoroacetylamines, N-trifluoroacetylamino acid esters and α-methyl- and α-phenylcarboxylic acid amides. The best efficiency is obtained when R′ is aromatic, particularly α-naphthyl, as in N-lauroyl-S-α-(1-naphthyl)ethylamine. The highest resolution factors were found for aromatic solutes, such as N-trifluoroacetyl-α-phenylethylamine and α-phenylbutyric acid amides, which could be resolved readily on packed columns. The enantiomers of α-branched carboxylic acids were separated for the first time by gas chromatography. Based on the packing arrangement in the crystalline form of the N-acetyl homologue of N-lauroyl-S-α-(1-naphthyl)ethylamine, a mechanism for the resolution is proposed. It is assumed that the mode of association found in the solid state is at least partially retained in the melt through a network of hydrogen bonds. The mechanism proposed is developed with particular reference to the aromatic solute-solvent systems. It is suggested that the solute is intercalated (“sandwiched”) between two solvent molecules. Arguments, based on X-ray data, are given to explain the selectivity observed.

Resolution of optical isomers by thin layer chromatography

Abstract A process for the separation of enantiomers by TLC is described. Reversed-phase plates, pre-treated with a copper II complex of N,N-di-n-propyl-L-alanine separate all the dansyl protein amino acids, except proline, each to its D and L enantiomers.

Three-dimensional image reconstruction from ordered arrays of 70S ribosomes

A better understanding of the molecular mechanism of protein biosynthesis still awaits a reliable model for the ribosomal particle. We describe here the application of a diffraction technique, namely three-dimensional image reconstruction from two-dimensional sheets of 70S ribosomes from Bacillus stearothermophilus at 47 A resolution. The three-dimensional model obtained by these studies shows clearly the two subunits, the contact points between them, an empty space large enough to accommodate the components of protein biosynthesis, the location of regions rich in RNA and a possible binding site for mRNA. The tunnel within the 50S particle which may provide the path taken by the nascent polypeptide chain in partially resolved.

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.

Ribosomal crystallography : from crystal growth to initial phasing

Preliminary phases were determined by the application of the isomorphous replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosomal subunits from halophilic and thermophilic bacteria. Derivatization was performed with dense heavy atom clusters, either by soaking or by specific covalent binding prior to the crystallization. The resulting initial electron density maps contain features comparable in size to those expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron microscopy in positively stained thin sections of embedded three-dimensional as well as with phase sets obtained by ab-initio computations. Aimed at higher resolution phasing, procedures are being developed for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by mutagenesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophil!c ribosomal subunits which yield crystals diffracting to the highest resolution obtained so far for ribosomes, 2.9 and 7.3 A, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosomal proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cysteines) and overexpressed. To assist the interpretation of the anticipated electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom binding and for attempting the determination of their three-dimensional structure, their components are being produced genetically. The low resolution models reconstructed from tilt series of crystalline arrays of ribosomal particles are being employed for initial phasing. The tentative functional interpretation of these models stimulated the design and the crystallization of complexes mimicking

The identification of selected components in electron density maps of prokaryotic ribosomes at 7 Å resolution

Crystals of small and large ribosomal subunits from thermophilic and halophilic bacteria, diffracting to 3 A, are being subjected to structural analysis with synchrotron radiation. The bright beam necessary for detecting and collecting the diffraction at the higher-resolution shell causes significant decay even at 25 K. Nevertheless, data collected from native and heavy-atom-derivatized crystals led to the construction of electron density maps of both ribosomal subunits, showing recognizable morphologies and internal features similar to those observed by EM reconstructions of the corresponding ribosomal particle. The main features of these maps include elongated dense regions traceable as well separated RNA duplexes or single strands. Also seen are globular patches of lower density, readily distinguishable from the above, in which folds observed by NMR or crystallography in isolated ribosomal proteins at atomic resolution were detected. The intercomponents contacts identified so far reveal diverse modes of recognition. Metal clusters, attached at selected sites on the particles, are being exploited to facilitate unbiased map interpretation. In this way, two surface proteins were located and several surface RNA strands were targeted.

Crystallography of ribosomes: Attempts at decorating the ribosomal surface

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Genetic and biochemical manipulations of the small ribosomal subunit from Thermus thermophilus HB8.

Abstract Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3Å and exhibit reasonable isomorphism and moderate resistance to irradiation. A 5Å MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs. To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing. We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means. These proteins can be modified and subsequently reconstituted into depleted ribosomal cores. They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them. In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.

Crystallographic, biochemical and genetic studies on halophilic ribosomes

Summary Ribosomal particles from Haloarcula marismortui were crystallized. The best crystals, diffracting to 2.9 A resolution and yielding crystallographic data of reasonable quality were grown from the large ribosomal subunits. Attempts at crystallization of functional ribosomal complexes are in progress, benefiting from experience gained from crystals of ribosomes of an extreme thermophilic bacterium, Thermus thermophilus . For obtaining phase information, a monofunctional reagent was prepared from an undecagold cluster, by attaching to it a chemically reactive handle, specific for sulfhydryl moieties. Heavy atom derivatives were prepared by binding this cluster to exposed sulfhydryls prior to the crystallization. Cores of halophilic ribosomal particles, lacking four ribosomal proteins, were prepared using dioxane. All detached proteins could be fully reconstituted. However, blocking the -SH group of one of them (HmaL11), prevented its incorporation into the core particles. The so obtained depleted 50S subunits crystallize under the same conditions as native ones and show apparent isomorphism with them. Most of the genes of several r-proteins were cloned. These are being used for sequencing as well as for providing new locations for binding heavy atom clusters by genetic insertion of cysteines on the ribosomal surface, according to accessibility data, obtained either chemically or by limited proteolysis. A nucleoprotein complex of protein HmaL1 and a fragment of the 23S rRNA was isolated from ribosomes. Chimeric complexes were reconstituted with E. coli ribosomal components, indicating rather high homolgy, despite the evolution distance.