Synthesis of a radioactive labeled undecagoldcluster for application in X-ray structure analysis of ribosomes.

In order to quantitate and to determine the extent of binding of a heavy atom derivative to ribosomal particles and other components of protein biosynthesis, we prepared a 14C-radioactive labeled monofunctional undecagoldcluster. This cluster can be bound either to exposed sulfhydryl groups at the surface of the ribosomes and its subunits, or to high nucleophilic amino groups of nucleic acids as well as to proteins.

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.

Low-resolution models for ribosomal particles reconstructed from electron micrographs of tilted two-dimensional sheets.

Models of the whole ribosome (70S) and its large subunit (50S) were obtained at low resolution (47 and 28 A respectively) by three-dimensional image reconstruction using diffraction data collected from electron micrographs of two-dimensional ordered arrays. The comparison of the various reconstructed images, using interactive computer graphics, enabled the assessment of the reliability of the method, the derivation of the shape of the small subunit (30S) and the assignment of several functional features such as the probable path taken by the nascent protein chain, the presumed site for the process of biosynthesis of proteins, and a feasible mode for tRNA binding. The reconstructed models of the various ribosomal particles may be used for phasing of X-ray diffraction data at low resolution.

Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies.

A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source. Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes. In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes. The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay. Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites.

Novel procedures for derivatization of ribosomes for crystallographic studies.

Undecagold and tetrairidium clusters have been used for the preparation of heavy-metal derivatives of ribosomal particles, necessary for the evaluation of phases in the x-ray structure determination of these large particles. To obtain specific binding, monofunctional reagents of the clusters were prepared and were covalently bound to free sulfhydryl groups on the surface of the ribosome. In addition, a mutant of Bacillus stearothermophilus which lacks one ribosomal protein (BL11) was grown. The heavy-atom clusters were covalently bound to isolated protein BL11, and the modified protein was consequently reconstituted into the mutated ribosomal subunits. Crystallographic data have been collected from crystals of native particles, from the mutated ones, and from the iridium- and gold-derivatized subunits. All these crystal forms are isomorphous within the experimental error.

Challenging the three-dimensional structure of ribosomes.

A prerequisite for understanding the detailed mechanism of the intricate process of protein biosynthesis is the determination of the molecular structure of ribosomes. This may be obtained by crystallographic analysis. Although such studies, in general, provide static pictures, they do indicate how to design subsequent functional and dynamic experiments. Thus, investigating models obtained by three-dimensional image reconstruction have stimulated further biochemical as well as crystallographic studies.

Cryocrystallography of ribosomal particles.

Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains. At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes. However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated. Hence it has become possible to collect a complete data set from one single crystal. Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane. Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity. In several cases soaking in a cryosolvent was required. Crystallographic data were collected with intense synchrotron radiation. Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H. marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B. stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T. thermophilus.

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis.

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.

Characterization of crystals of small ribosomal subunits.

Crystals of intact small ribosomal subunits from Thermus thermophilus have been obtained from functionally active particles. The crystals (P42(1)2, 407 A x 407 A x 171 A) are suitable for X-ray crystallography analysis to 9.9 A using synchrotron radiation at cryotemperature. Crystallographic data from native and a potential heavy-atom derivative have been collected.

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal subunits containing a nascent polypeptide chain.

Polylysine chains were synthesized on Bacillus stearothermophilus ribosomes in a poly(A)-programmed in vitro system. After separation of the ribosomal subunits by sucrose gradient centrifugation, the polylysine chains (in contrast to the polyphenylalanine chains synthesized in a poly(U) system) reproducibly remained attached to the large ribosomal subunit. It was possible to produce two-dimensional crystalline sheets from the large ribosomal subunits containing the polylysine chains. These sheets are an essential prerequisite for three-dimensional reconstruction studies aiming to show that the tunnel in the large ribosomal subunit provides a path for the nascent polypeptide chain.

Approaching the molecular structure of ribosomes.

Fifteen forms of three-dimensional crystals and three forms of two-dimensional sheets from ribosomal particles have been grown. In all cases only biologically active particles could be crystallized, the crystalline material retaining its integrity and biological activity for months. Cryastallographic data have been collected from crystals of 50 S ribosomal subunits, using synchrotron radiation, at temperatures between 19 and -180 degree C. Although at around 0 degrees C in the synchrotron X-ray beam the crystals rapidly lose their high-resolution reflections, at cryo-temperatures hardly any radiation damage occurs over long periods, and a complete set of diffraction data to about 6 A resolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50 S ribosomal subunits from a mutant of Bacillus stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with the native form. Models of the entire 70 S ribosome and of the 50 S subunit have been reconstructed from two-dimensional sheets at 47 and 30 A, respectively. These models demonstrate the overall shape of the particles, the contact areas between large and small subunits, the space where protein biosynthesis may take place and a tunnel through the 50 S subunit which could provide a path for the nascent polypeptide chain.

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures.

A procedure for the in vitro growth of well-ordered two-dimensional sheets from ribosomal particles using salts and salt-alcohol mixtures has been developed. Employing this procedure, ordered two-dimensional sheets of the wild type as well as of mutated 50 S ribosomal subunits from Bacillus stearothermophilus can readily be obtained. These sheets, stained with uranyl acetate or gold-thioglucose, are suitable for three-dimensional image reconstruction. They consist of relatively small unit cells with dimensions of 160 +/- 15 and 365 +/- 20 A. Diffraction patterns of electron micrographs of these sheets contain features to 25 A resolution.

Reconstitution and crystallisation experiments with isolated split proteins from Bacillus stearothermophilus ribosomes.

Six proteins (B-L1, B-L6, B-L10, B-L11, B-L12 and B-L16) were removed from 50S ribosomal subunits of Bacillus stearothermophilus by treatment with ethanol and ammonium chloride. The proteins were isolated in a pure form, and one of them (B-L6) was crystallized. Five of the six proteins (in various combinations) were added back to the core particles, resulting in 50S subunits lacking one protein. The biological activities of these ribosomal particles as determined in the poly(U)-system varied over a wide range, depending on the protein which was omitted. The particles lacking one protein provide useful tools for heavy-atom derivation necessary for our crystallographic studies on the 50S subunits of Bacillus stearothermophilus.

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.

A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction.

A better understanding of the molecular mechanism of protein biosynthesis depends on the availability of a reliable model for the ribosome particle. The application of a diffraction technique, namely, three-dimensional image reconstruction from two-dimensional sheets of the large ribosomal subunits of Bacillus stearothermophilus at a resolution of 30 angstroms is described. The resulting three-dimensional model shows at least four projecting arms, arranged radially near the presumed interface with the 30S subunit. The projecting arms are positioned around a cleft, which turns into a tunnel with a length of 100 to 120 angstroms and a diameter of up to 25 angstroms. This tunnel spans the particle and may provide the path taken by the nascent polypeptide chain.

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A.

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.