M. Shoham

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

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.

Crystal structure of demetallized concanavalin A: the metal-binding region.

Abstract We have determined the crystal structure of demetallized concanavalin A, at a resolution of 3.2 A, by molecular replacement using the known structure of native concanavalin A. Refinement of the initial model using a constraint-restraint reciprocal-space least-squares procedure caused the conventional crystallographic agreement ( R ) factor to decrease from 0.47 to a final value of 0.26. There are significant conformational changes in the metal-binding region involving residues Asp 19 and His24, which are substantially closer to each other than in native concanavalin A. These residues form an internal salt bridge which does not exist when the metal ions are attached to the protein. The binding site for transitionmetal ions is still intact, but the calcium site is not, since one of its two carboxylic ligands, Asp 19, is unavailable. Flexibility is observed for one of the transitionmetal ligands, Glu8, as well as for some segments of the backbone. The latter could account for the increased susceptibility of demetallizcd concanavalin A to proteolysis.

Crystallography of ribosomal particles

Abstract Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Aresolution 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 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30A, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

Crystallization of halophilic malate dehydrogenase from Halobacterium marismortui

Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group 12 with cell dimensions a = 113.8 A, b = 122.8 A, c = 126.7 A, beta = 98.1 degrees. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions a = 118.6 A, b = 88.9 A, c = 165.9 A, beta = 103.0 degrees and one tetramer of 120,000 Mr per asymmetric unit. The crystals are suitable for high-resolution work.

A new crystal form of large ribosomal subunits from Halobacterium marismortui

A new form of three‐dimensional crystals of the 50 S ribosomal subunits from Halobacterium marismortui has been obtained at 19°C, using polyethylene glycol in the presence of 1.2–1.7 M KCl in the crystallization mixture. The crystals diffract X‐rays to 13 Å and are stable in the synchrotron radiation beam for 4–8 h. Being aggregates of thin plates, the dimensions of only two unit cell edges, 147 × 181 Å, with an angle of 95°, could so far be determined by both X‐ray crystallography and electron microscopy. Attempts to produce thicker crystals by sophisticated seeding are in progress.

The effect of binding of metal ions on the three‐dimensional structure of demetallized concanavalin A

Concanavalin A (con A) is a saccharide-binding protein of the Jack bean [ 11. The protein contains two different metal-binding sites, which must be occupied in order for saccharide binding to be possible [2,3]. When the metal ions are removed by acid treatment, the demetallized protein can bind only divalent transition metal ions (Mn”, Co”, Ni2+, Zn”, or Cd23 at a site designated S 1, but it cannot bind Ca2+ or saccharide. When Sl is occupied by one of the metal ions listed above, the protein can bind Ca” at a second metalbinding site designated S2 [4]. We have made an X-ray crystallographic determination of the three-dimensional structure of demetallized concanavalin A and we report here the important structural differences in the metal-binding region between the demetallized protein and the native metalloprotein [5-71 (fig.1). We postulate a detailed mechanism whereby binding of the transition metal ion at Sl engenders the Ca2+-binding site (S2). The experimental details, refinement procedure and electron density maps upon which our structure is based will be reported fully [8].

Purification and characterization of ribosomal proteins from the 30 S subunit of the extreme halophile Halobacterium marismortui

Ribosomal proteins were extracted from 30 S subunits of Halobacterium marismortui under native conditions. Their separation was based on gel filtration and hydrophobic chromatography, performed at a concentration of 3.2 M KCl to avoid denaturation. A total of nine proteins were isolated, purified and identified by partial amino‐terminal sequences and two‐dimensional gel electrophoresis. There is a high degree of sequence homology with 30 S proteins from H. cutirubrum, and also some with 30 S proteins of eubacteria. Proton NMR data indicate unfolding of the proteins in low salt. One of the proteins, however, retains its secondary structure at a salt concentration as low as 0.1 M NaCl, and even in 8 M urea. One reason for this outstanding stability could be the high proportion (50%) of β‐structure in this protein as determined from circular dichroism measurements. In general, there is a higher β‐sheet content than for 30 S proteins from Escherichia coli. Measurements of Stokes radii indicate several of the proteins to have a rather elongated shape. One of these is a complex consisting of L3/L4 and L20, similar to the L8‐complex from E. coli. The presence of this 50 S complex in the preparation of the small subunit suggests a location on the interface between the subunits.

Crystallization and preliminary X-ray diffraction studies of colicin E3 immunity protein

Immunity protein, an inhibitor of the ribonuclease activity of the protein antibiotic colicin E3, crystallizes in the orthorhombic space group C222 with cell dimensions a = 78.7 A, b = 54.1 A, c = 36.1 A and one molecule of Mr 9800 per asymmetric unit. The crystals are suitable for high resolution X-ray analysis.

Comparison of different density modification methods for improving the image of a protein map at high resolution

A symmetry-averaging procedure in which electron density values to be averaged are retrieved, and averages inserted, by means of a superposed symmetry grid (Nordman (1980), Acta Cryst. A36, 747-754) was used in the lowresolution structure analysis of satellite tobacco necrosis virus (STNV), where 10 ~ phases from double isomorphous replacement (DIR) were used as the starting point in a stepwise phase extension to 4.0 A (Unge et al. (1980), Nature 285,373-377). To evaluatetheprocedure in a case less extreme (i.e., less favorable) than the 60-fold noncrystallographically symmetric STNV, we have applied an improved version to hemocyanin, \-lith 6-fold noncrystallographic symmetry (Gaykema et~. (1983), 0 Chemica Scripta 21,19-23) using IFobs!values to 3.2 A resolution and DIR phases to 5.0 ~ kindly provided by W. G. J. Hol, University of Groningen.