Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution.

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the alpha-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.

A synchrotron x-ray diffraction study of developing chick corneas.

To study some ultrastructural aspects of developing chick corneas we performed a synchrotron x-ray diffraction analysis of 22 specimens obtained daily from developmental day 10 through day 19. Before day 12 of development in chicks we were unable to detect a meridional x-ray diffraction pattern from cornea. Neither were we able to record a first-order equatorial x-ray reflection at this time. Normally, these reflections are present in corneal x-ray patterns, arising from, respectively, the periodic axial electron density of fibrillar collagen and the lattice-like arrangement of the fibrils. By day 12 of development we could detect the third- and fifth-order meridional reflections (indicating increased amounts of collagen) and a first-order equatorial reflection (implying that more collagen was regularly arranged). The third- and fifth-order meridional reflections became more intense as the tissue matured, suggestive of a continued deposition of fibrillar collagen, and the scattering angle of the interfibrillar maximum increased, suggesting that regularly arranged collagen was becoming more closely packed with maturation. In embryonic chick corneas, the establishment of an orderly, fairly compacted matrix of collagen fibrils may be one of the main events underlying the acquisition of corneal transparency.

Macular corneal dystrophy type II: multiple studies on a cornea with low levels of sulphated keratan sulphate.

We investigated an individual macular corneal dystrophy (MCD) type II cornea from a 42-year-old woman with markedly reduced antigenic keratan sulphate levels. A characteristic 4.6 A X-ray reflection was evident, and the mid-stroma contained 30% less sulphur than normal. Close packing of collagen was restricted to the superficial stroma. Abnormally large proteoglycan filaments were noted throughout the extracellular matrix and Descemet's membrane's posterior non-banded zone, but not its anterior banded zone. Small, collagen-associated stromal proteoglycans were susceptible to digestion with chondroitinase ABC, but not keratanase I or N-glycanase. On occasion, collagen fibrils ranged in size from 20 nm to 58 nm, with preferential diameters of 34 nm and 42 nm. Corneal guttae were evident, as were numerous endothelial inclusions, most probably due to intracellular fibrillogranular vacuoles similar to those found in the stroma. The endothelium expressed reduced anti-keratan sulphate labelling.

Proteoglycans contain a 4.6 A repeat in muscular dystrophy corneas: x-ray diffraction evidence.

Synchrotron x-ray diffraction patterns from macular corneal dystrophy (MCD) corneas contain an unusual reflection that arises because of an undefined ultrastructure with a periodic repeat in the region of 4.6 A. In this study, we compared with wide-angle x-ray diffraction patterns obtained from four normal human corneas and four MCD corneas. Moreover, portions of two of the MCD corneas were pretreated with a specific glycosidase to shed light on the origin of the 4.6 A reflection. None of the normal corneas produced an x-ray reflection in the region of 4.6 A, whereas all four of the MCD corneas did (MCD type I at 4.65 A and 4.63 A, MCD type II at 4.63 A and 4.67 A). This reflection was diminished after incubation of the MCD tissues with either chondroitinase ABC or N-glycanase. The findings indicate that glycosaminoglycans or proteoglycans contribute to the unusual MCD x-ray reflection and hence most likely contain a periodic 4.6 A ultrastructure. Furthermore, the results imply that periodic 4.6 A MCD ultrastructures reside in either intact, unsulfated lumican molecules and regions of the CS/DS-containing molecules or in a region of a hybrid macromolecular aggregate formed by the interaction of the two molecules.

Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetradecamer with an axial pore.

The Escherichia coli ATP-dependent caseinolytic protease (Clp) is composed of two distinct subunits; protease, ClpP, and ATPase, ClpA. Active ClpP has been overexpressed to approximately 50% of soluble protein in E. coli, and purified to homogeneity. Direct mass determination of individual particles using scanning transmission electron microscopy (STEM) yields a mean native molecular mass of 305 +/- 9 kDa for the ClpP oligomer, suggesting that it has a tetradecameric structure. Small-angle X-ray scattering (SAXS) curves were determined for ClpP in solution at concentrations of 1-10 mg/mL. A combination of STEM and SAXS data was used to derive a model for ClpP, comprising a cylindrical oligomer about 100 A in diameter and about 75 A in height with an axial pore about 32-36 A in diameter. The volume of the pore is estimated to be approximately 70,000 A3, similar in size to those found in chaperone proteins, and is large enough to accommodate unfolded polypeptide chains, although most globular folded proteins would be excluded.

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli.

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

A role for proteins S3 and S14 in the 30 S ribosomal subunit.

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.

Characterization of Nicotiana tabacum chloroplast and cytoplasmic ribosomal proteins.

Proteins from Nicotiana tabacum cytoplasmic and chloroplast ribosomes and their subunits have been isolated under a variety of conditions and resolved by two-dimensional polyacrylamide gel electrophoresis. Average absolute mobility maps, constructed from the resultant electropherograms, were used to compare ribosomal proteins from cytoplasmic and chloroplast ribosomes. A novel technique for the estimation of molecular weights from two-dimensional electrophoretic mobilities is described. The cytoplasmic ribosome of N. tabacum possesses 73-80 distinct proteins with pI greater than 5, 26-30 associated with the 40 S subunit, and 47-50 with the 60 S subunit. The 60 S cytoplasmic ribosomal subunit has, in addition, at least three acidic polypeptides (pI less than 5). The chloroplast ribosome has 55-58 unique basic proteins with 22-23 occurring in the 30 S subunit and 33-35 in the 50 S subunit. A few additional acidic polypeptides are associated with the 30 S and 50 S subunits (2-3 and 1, respectively). There is little similarity between the electrophoretic patterns or molecular weight frequency distributions of proteins of analogous cytoplasmic and chloroplast ribosomal subunits of N. tabacum. The electrophoretic patterns and molecular weight frequency distributions of the proteins of N. tabacum chloroplast ribosomal subunits are quite similar to those of the Escherichia coli ribosome and the chloroplast ribosome of the alga Chlamydomonas. N. tabacum cytoplasmic ribosomal protein electrophoretic patterns and molecular weights are very similar to proteins from the Chlamydomonas cytoplasmic ribosome. These data substantiate: 1) the close affiliation between higher plant chloroplast and prokaryotic ribosomes and 2) a general similarity between angiosperm cytoplasmic ribosomal proteins and other classes of eukaryotic cytoplasmic ribosomal proteins.