Crystals of threonyl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.     

Crystals of seryl-tRNA synthetase from Escherichia coli. Preliminary crystallographic data

Crystals of seryl-tRNA synthetase from Escherichia coli can be grown from ammonium sulphate/octyl glucoside solutions in two days. The crystals appear to be very suitable for X-ray analysis, diffracting to at least 2.8 A resolution and being resistant to radiation damage. The crystals are monoclinic (space group C2) with cell parameters a = 148.2 A, b = 90.6 A, c = 69.5 A and beta = 119.0 degrees. Depending on whether the asymmetric unit is the enzyme monomer (Mr 48,414) or dimer the Vm value would be either 4.12 or 2.10 A3/dalton. Although the former would indicate a rather high solvent content, other proteins crystallized in the presence of octyl glucoside have Vm values similar to this.     

Preliminary crystallographic study of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been isolated and crystallized. The enzyme was found to consist of two types of subunits with molecular masses 38 X 10(3) (alpha) and 94 X 10(3) (beta) and is likely to be a tetrameric protein with a molecular mass of about 260 X 10(3) (alpha 2 beta 2). Crystals of phenylalanyl-tRNA synthetase were grown by the hanging-drop technique at 4 degrees C in the presence of ammonium sulfate. Trigonal crystals, space group P3(1)21, with cell dimensions a = b = 176 A and c = 142 A (1 A = 0.1 nm), are suitable for medium-resolution X-ray analysis.     

Crystals of protein L1 from the 50 S ribosomal subunit of Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.     

Thermus thermophilus ribosomes for crystallographic studies.

Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies.     

Preliminary crystallographic data for adenylosuccinate synthetase from rabbit skeletal muscle

Single crystals of adenylosuccinate synthetase, an essential component of purine biosynthesis, extracted from rabbit skeletal muscle were prepared as suitable specimens for X-ray structure analysis. The crystal belongs to the space group P4₃2₁2 or P4₁2₁2 with unit cell dimensions $\text{a = b = 71.2}\text{A}\text{?}\text{, c = 194.8}\text{A}\text{?}$. The asymmetric unit contains one protein molecule of 54,000 molecular weight.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

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.     

Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8

Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 ₁22 (or P6 ₅22), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of ribosomal protein S8 from Thermus thermophilus

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2-methyl-2,4-pentandiol (v/v). The crystals belong to the space group P4₁₍₃₎2₁2 with cell parameters a= b= 67.65 Å, c= 171.12 Å. They diffract x-rays to 2.9 Å resolution. © 1997 Wiley-Liss, Inc.     

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of - and β-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native -subunits of both enzymes were primarily ₂-dimers and tended to aggregate. Most E. coli enzyme β-subunits were monomeric and only a small fraction was represented by β₂-dimers. All thermophilic β-subunits were β-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA^Phe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β-subunits. -Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.     

Crystals of intact elongation factor Tu from Thermus thermophilus diffracting to high resolution

The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution. They exhibit space group C2, with a = 150.3(6) A, b = 99.6(3) A, c = 40.1(1) A, beta = 95.4(2) degrees, and contain one elongation factor Tu molecule per asymmetric unit.     

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.     

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.     

Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.     

Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus

Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.