Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data.

The glycyl-tRNA synthetase from Thermus thermophilus is a dimer of molecular mass 115 kDa, which has been crystallised using the vapour diffusion method from 5 to 7% polyethylene glycol 6000, 0.8 to 1.4 M NaCl at protein concentrations of 2 to 8 mg/ml. Nucleation is carried out at 4 degrees C and crystals are subsequently transferred to 15 degrees C to maximise growth. Crystals are truncated rhombohedra measuring on average 0.4 mm x 0.4 mm x 0.2 mm, which appear within a few days and reach full size in one to two months. GlyRS crystallises in two closely related space groups, P2(1)2(1)2(1) and C2,2,2(1), both with the same cell a = 125 A, b = 254 A, c = 104 A. Crystal packing in P2(1)2(1)2(1) is strongly C-centred. The crystals have VM = 3.6 A3/Da and a solvent content of 61%, with one dimer in the asymmetric unit in C2,2,2(1) and two dimers in P2(1)2(1)2(1). The best native data extend to 2.9 A in C2,2,2(1) and are 90.6% complete with an R-factor between symmetry-related reflections of 10.0%. The structure has been solved by multiple isomorphous replacement and model building is in progress.

Crystallization of threonyl-tRNA synthetase from Thermus thermophilus and preliminary crystallographic data.

Threonyl-tRNA synthetase from Thermus thermophilus (ttTRS) has been overproduced in Escherichia coli, purified and crystallized in solutions containing ammonium sulfate and glycerol. The crystals grew in the orthorhombic space group C222(1) with unit cell dimensions a = 119.5 A, b = 120.0 A, c = 317.5 A. The asymmetric unit is constituted of two monomers and the crystals contain 66% solvent. This paper reports the first crystals of ttTRS and preliminary crystallographic results since the presumed crystals of ttTRS described in a previous paper [1] were crystals of aspartyl-tRNA synthetase [2].

Heavy-atom refinement against solvent-flattened phases.

A new algorithm for refinement of heavy-atom parameters is defined by an iterative procedure where external phases are provided by density modification. This algorithm is applied to two cases, tRNA(Asp) and the complex between tRNA(Asp) and aspartyl-tRNA synthetase. In the first case, where the structure was solved by multiple isomorphous replacement (MIR) methods, it was found that the new method gives accurate values for the native-derivative scale and four occupancy of heavy-atom sites. Position refinement was more delicate and it needed to be handled in a restricted resolution range. In the second case, where a similar method was used in the early stages of solving the phase problem, it slightly decreased the phase error. It was followed by an improvement of the density-modification masks, which led to better maps at higher resolution.

Sequence analysis and modular organization of threonyl-tRNA synthetase from Thermus thermophilus and its interrelation with threonyl-tRNA synthetases of other origins.

The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodon-binding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity. Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.