J. Mol. Biol. (1990) 214, 819-820
Crystals of Threonyl-tRNA Synthetase from Thermus thermophilus Preliminary Crystallographic
Data
M. B. Garber’, A. D. Yaremchk2, M. A. Tukhlo2, S. P. Egorova2 N. P. Fomenkoval and S. V. Nikonovl ‘Institute of Protein Research Academy of Sciences of the U.S.S.R. 142292 Pushchino, Moscow Region, U.S.S.R. 21nstitute of Molecular Biology and Genetics Academy of Sciences of the Ukrainian S.S.R., Kiev, U.S.S.R. (Received 1 May 1990; accepted 1 May 1990) 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 P212,2, with cell parameters a=61.4 A, b= 156.1 A, c= 177.3 A.
been described from T. thermophilus has (Yaremchuk et al., 1989). The isolated enzyme is functional in aminoacylating cognate tRNA from E. coli. Like the E. coli enzyme: it is composed of two identical subunits, (CI~ type) with MI of about 2 x 74,000. Determination of the primary structure of the enzyme has been started. Crystallization trials were carried out using the hanging drop/vapour diffusion technique with 10 ~1 drops of protein solution equilibrated against 500 ~1 of precipitant solution. Crystals were obtained with citrate and sodium methylpentane diol, sodium formate. The best crystals grow with sodium formate as a precipitant. The protein solution (approx. 10 mg/ml) was dialysed against 50% saturated sodium formate in 50 mivr-Tris . HCl (pH 7.5), 5 mM-dithiothreitol, 5 mM-MgCl,. The amorphous precipitate obtained after dialysis dissolves in the drops equilibrated against 40 y0 saturated sodium formate and the crystals grow for one to two weeks to 600 pm x 300 pm x 150pm at 4°C. These crystals, after washing with 53% saturated sodium formate (pH 7.5), were examined by SDS/polyacrylamide gel electrophoresis; only a single band with a mobility corresponding to the original enzyme was found on Coomassie blue staining. X-ray diffraction measurements were carried out on an Elliott GX-13 rotating anode generator operated at 35 kV and 35 mA. The size of the focal spot
Determination of spatial structures of aminoacyltRNA synthetases is an important part of proteinsynthesizing system investigations. Several bacterial aminoacyl-tRNA synthetases and one from yeast have been crystallized and are now being investigated by X-ray analysis. The crystal structures of two aminoacyl-tRNA synthetases, those of Bacillus stearothermophilus tyrosyl-tRNA synthetase (Brick et al., 1988) and Escherichia coli methionyl-tRNA synthetase (Brunie et al., 1987), have been determined as complexes with either ATP or amino acid substrates. The crystal structure of E. co&i glutaminyl-tRNA synthetase complexed with tRNAG1” and ATP has been determined at 2.8 A (1 A=O*l nm: Rould et al., 1989). The crystal structure of the complex between yeast tRNAASp and aspartyl-tRNA synthetase is in progress (Ruff et al., 1988). The extreme thermophilic bacterium Thermus thermophilus is a very convenient source for isolation and crystallization of many components of the protein-synthesizing system (Garber et al., unpublished results). At present, two crystalline aminoacyl-tRNA synthetases (phenylalanyland seryl-) from T. thermophilus have been studied (Chernaya et al., 1987; Garber et al., unpublished results). Here, we report the crystallization and preliminary crystallographic data of threonyl-tRNA synthetase from this micro-organism. Purification of the threonyl-tRNA synthetase 0022-2836/90/16081942
$03.00/O
819
0
1990 Academic
Press Limited
820
i!!l.
B.
Chrber et ai. -
was 0.1 x 0.2 mm2. Precession photographs were taken with an Enraf-Nonius camera. The crystal-tofilm distance was 75 mm. Analysis of the diffraction pattern symmetry and unit cell pa,rameters was done from precession photographs of the main zones at about 5 !L resolution. From these photographs, it was established that t,he crystals belong to the space group P2,2,2, and that t)he unit cell parameters are a=61.4 8, b= 156.1 8, c= 177.3 ,JJ. The unit cell volume is 1.70 x IO6 A3 with probably a dimer as the asymmetric unit and four dimers per unit cell. This is most probable, since a calculation of the volume packing parameters V, according to Matthews (1968) gives a value of 3.17 A3/dalton for four dimers and the unlikely value of 1.58 a3/dalton for eight dimers per unit cell. On still photographs, the diffraction pattern extends to 2.4 a and the crystals displayed this diffraction limit during 100 hours at room temperature.
This work was stimulated by Professor M. Orunberg-Manago. who has investigated the structure and functions of threonyl-tRNA synthetase from E. coli very intensively for msny years.
References Brick, P., Bhat; T. h‘.. Blow, B. 31. (1988). J. Xoi. Viol. 208, 83398. Brunie, S., Mellot, P., Ze!wer, C.. Risler. J. I,.; Blancjuec. S. & Fayat, G. (1987). j. Xol. Graph. 5; 1821. Chernaya, M. XI., Iiorolev, S. V., Reshetnikova,. L, S. & Safro. M. G. (1987). J. Xol. Biol. 198. 555-556. Matthews, B. W. (1968). J. Mol. Biol. 33. 491-497. Rould, Xl. A.. Perona, J. !I., Soll, 1). & Steitq T. A. (I 989). Scimce, 246, 1135-1191. Ruff, M., Cavareili, J., Mikol, V., Losber, B., Mitschler. A., Giege, R.. Thierry. J. C. & Moras, 11. (1988). J. MOT. Bid. 261, 236-236. Yaremchuk, A. D., Tukalo, MCI.A., Egorova. S. I’. B Xat,suka, G. Kh. (1989). Riopol. Kletka (U.S.S.R,), 5. 102-104.
Edited by A. Khg
Crystals of Threonyl-tRNA Synthetase from Thermus thermophilus Preliminary Crystallographic
Data
M. B. Garber’, A. D. Yaremchk2, M. A. Tukhlo2, S. P. Egorova2 N. P. Fomenkoval and S. V. Nikonovl ‘Institute of Protein Research Academy of Sciences of the U.S.S.R. 142292 Pushchino, Moscow Region, U.S.S.R. 21nstitute of Molecular Biology and Genetics Academy of Sciences of the Ukrainian S.S.R., Kiev, U.S.S.R. (Received 1 May 1990; accepted 1 May 1990) 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 P212,2, with cell parameters a=61.4 A, b= 156.1 A, c= 177.3 A.
been described from T. thermophilus has (Yaremchuk et al., 1989). The isolated enzyme is functional in aminoacylating cognate tRNA from E. coli. Like the E. coli enzyme: it is composed of two identical subunits, (CI~ type) with MI of about 2 x 74,000. Determination of the primary structure of the enzyme has been started. Crystallization trials were carried out using the hanging drop/vapour diffusion technique with 10 ~1 drops of protein solution equilibrated against 500 ~1 of precipitant solution. Crystals were obtained with citrate and sodium methylpentane diol, sodium formate. The best crystals grow with sodium formate as a precipitant. The protein solution (approx. 10 mg/ml) was dialysed against 50% saturated sodium formate in 50 mivr-Tris . HCl (pH 7.5), 5 mM-dithiothreitol, 5 mM-MgCl,. The amorphous precipitate obtained after dialysis dissolves in the drops equilibrated against 40 y0 saturated sodium formate and the crystals grow for one to two weeks to 600 pm x 300 pm x 150pm at 4°C. These crystals, after washing with 53% saturated sodium formate (pH 7.5), were examined by SDS/polyacrylamide gel electrophoresis; only a single band with a mobility corresponding to the original enzyme was found on Coomassie blue staining. X-ray diffraction measurements were carried out on an Elliott GX-13 rotating anode generator operated at 35 kV and 35 mA. The size of the focal spot
Determination of spatial structures of aminoacyltRNA synthetases is an important part of proteinsynthesizing system investigations. Several bacterial aminoacyl-tRNA synthetases and one from yeast have been crystallized and are now being investigated by X-ray analysis. The crystal structures of two aminoacyl-tRNA synthetases, those of Bacillus stearothermophilus tyrosyl-tRNA synthetase (Brick et al., 1988) and Escherichia coli methionyl-tRNA synthetase (Brunie et al., 1987), have been determined as complexes with either ATP or amino acid substrates. The crystal structure of E. co&i glutaminyl-tRNA synthetase complexed with tRNAG1” and ATP has been determined at 2.8 A (1 A=O*l nm: Rould et al., 1989). The crystal structure of the complex between yeast tRNAASp and aspartyl-tRNA synthetase is in progress (Ruff et al., 1988). The extreme thermophilic bacterium Thermus thermophilus is a very convenient source for isolation and crystallization of many components of the protein-synthesizing system (Garber et al., unpublished results). At present, two crystalline aminoacyl-tRNA synthetases (phenylalanyland seryl-) from T. thermophilus have been studied (Chernaya et al., 1987; Garber et al., unpublished results). Here, we report the crystallization and preliminary crystallographic data of threonyl-tRNA synthetase from this micro-organism. Purification of the threonyl-tRNA synthetase 0022-2836/90/16081942
$03.00/O
819
0
1990 Academic
Press Limited
820
i!!l.
B.
Chrber et ai. -
was 0.1 x 0.2 mm2. Precession photographs were taken with an Enraf-Nonius camera. The crystal-tofilm distance was 75 mm. Analysis of the diffraction pattern symmetry and unit cell pa,rameters was done from precession photographs of the main zones at about 5 !L resolution. From these photographs, it was established that t,he crystals belong to the space group P2,2,2, and that t)he unit cell parameters are a=61.4 8, b= 156.1 8, c= 177.3 ,JJ. The unit cell volume is 1.70 x IO6 A3 with probably a dimer as the asymmetric unit and four dimers per unit cell. This is most probable, since a calculation of the volume packing parameters V, according to Matthews (1968) gives a value of 3.17 A3/dalton for four dimers and the unlikely value of 1.58 a3/dalton for eight dimers per unit cell. On still photographs, the diffraction pattern extends to 2.4 a and the crystals displayed this diffraction limit during 100 hours at room temperature.
This work was stimulated by Professor M. Orunberg-Manago. who has investigated the structure and functions of threonyl-tRNA synthetase from E. coli very intensively for msny years.
References Brick, P., Bhat; T. h‘.. Blow, B. 31. (1988). J. Xoi. Viol. 208, 83398. Brunie, S., Mellot, P., Ze!wer, C.. Risler. J. I,.; Blancjuec. S. & Fayat, G. (1987). j. Xol. Graph. 5; 1821. Chernaya, M. XI., Iiorolev, S. V., Reshetnikova,. L, S. & Safro. M. G. (1987). J. Xol. Biol. 198. 555-556. Matthews, B. W. (1968). J. Mol. Biol. 33. 491-497. Rould, Xl. A.. Perona, J. !I., Soll, 1). & Steitq T. A. (I 989). Scimce, 246, 1135-1191. Ruff, M., Cavareili, J., Mikol, V., Losber, B., Mitschler. A., Giege, R.. Thierry. J. C. & Moras, 11. (1988). J. MOT. Bid. 261, 236-236. Yaremchuk, A. D., Tukalo, MCI.A., Egorova. S. I’. B Xat,suka, G. Kh. (1989). Riopol. Kletka (U.S.S.R,), 5. 102-104.
Edited by A. Khg
