J. Mol. Biol. (1990) 213, 631-632
Crystals of Seryl-tRNA Synthetase from Thermus thermophilus Preliminary Crystallographic Data M. B. Garber Institute of Protein Research, Academy of Sciences of the USSR 142292 Pushchino, Region, USSR
A. D. Yaremchuk, M. A. Tukalo, S. P. Egorova Institute of Molecular Biology and Genetics Academy oil Sciences of the Ukrainian SSR Kiev, USSR
C. Berthet-Colominas and R. Leberman E M B L Grenoble Outstation 156 X, 38042 Grenoble Cedex, France (Received 16 February 1990; accepted 6 March 1990) Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a=87"1 A, b= 126"9 A, c--63"5 A and fl= 109.7 °.
Crystallization trials were carried out using the hanging drop and vapour diffusion technique with 10-/~l drops of protein solution equilibrated against 500 #l of precipitant solution. Using ammonium sulphate solutions containing 5 mM-dithiothreitol 10 mM-MgCl2 1 mM-NaN3 (pH 6"2 to 8"0), very thin needle-like crystals were obtained. The morphology of these crystals was not improved by the addition of fl-octylglueoside. A variety of other additives were then tested, and it was found that the addition of methane pentane diol (MPDt) led to a marked improvement in crystal morphology. The final crystallization conditions were: drops containing 10 mg protein/ml and 20% saturated ammonium sulphate in 50 mM-Mes (pH 6"5), 5 mM-dithiothreitol, 10 mM-MgCt2, 1 mM-NaN3, 1% (v/v) MPD. These were equilibrated against 30 to 35% saturated ammonium sulphate containing 80/o MPD at room temperature, and crystals with dimensions of 0"3 mm x 0'5 m m x 1 mm could be obtained in a few days. These crystals, after washing with 50 % saturated ammonium sulphate solution containing 3%
Questions on the origin of thermostability of proteins might be answered by the study, at the atomic level, of the homologous protein from both mesophiles and thermophiles. Since in all organisms the aminoacyl-tRNA synthetases play an indispensable rSle in protein biosynthesis, the possibility of studying the same aminoacyl-tRNA synthetase from a mesophile and an extreme thermophile at the atomic level is an attractive proposition. The crystallization of seryl-tRNA synthetase from Escherichia coli has been described (Leberman et al., 1987) and a high-resolution structure of this enzyme is available (Cusack et al., unpublished results). We report the crystallization and preliminary crystallographic data of the homologous enzyme from the extreme thermophile Therrnus thermophilus. The purification of the seryl-tRNA synthetase (EC 6.1.1.11) from T. thermophilus has been described by Yaremchuk et al. (1989). The isolated enzyme is functional in aminoaeylating cognatetRNA from E. coll. Like the E. coli enzyme it is composed of two identical subunits but with a slightly smaller Mr value of about 46,000. The determination of the primary structure of the enzyme is currently underway. 0022-2836/90/120631-02 $03.00/0
Abbreviations used: MPD, methane pentane diol; Mes, 4-morpholino-ethanesulphonic acid. 631
~} 1990AcademicPress Limited
632
M . B . Garber et al.
MPD, were examined by SDS/polyacrylamide gel electrophoresis. A single band with a mobility corresponding to the original enzyme was found on Coomassie blue staining. The protein recovered from dissolved crystals also showed enzymic activity in the aminoacylation of tRNA. It should be noted that the enzyme is remarkably stable, retaining its integrity and biological activity after one year at room temperature under the crystallization conditions. A problem that arose was the handling of the crystals on mounting them in capillaries for X-ray diffraction studies in that using artificial mother liquor, they tended to dissolve. This problem was overcome by cross-linking with glutaraldehyde. Glutaraldehyde was added to the equilibration solution to a final concentration of 0"5% (v/v) once the crystals had grown, and they were left to equilibrate further for two days, then glutaraldehyde was added directly to the protein drop to a final concentration of 0"5~/o and left overnight. Crystals were then washed with a solution containing 30~o saturated ammonium sulphate and 2 ~/o MPD (pH 6"5). The cross-linked crystals were very stable and could be examined by X-ray diffraction. The crystals were mounted in glass capillaries and examined by CuKa X-radiation from a GX21 Elliot rotating anode generator using an Enraf-Nonius precession camera with a crystal-to-film distance of 60 mm. Unit cell dimensions were determined from l l ° precession photographs, by scanning the films with an Optronix rotating drum scanner (50 gm raster), followed by peak search and lattice refinement using subroutines of the SEMPER image-
processing package (Saxton et al., 1979). Crystals mounted with one edge parallel to the drum rotation axis gave precession photographs that showed cmm symmetry. This suggested that the apparent edge corresponds to a 2-fold axis; this was confirmed by the first upper level photograph that contained only the symmetry corresponding to the mirror perpendicular to the 2-fold axis, If the crystal was mounted with the same apparent edge parallel to the X-ray beam, then the precession photograph was of the hO1 plane. From precession photographs it was established that the crystals belong to the monoclinic space group P21, and that the unit cell parameters are a = 8 7 ' l A , b=126"9A, c = 6 3 . 5 A and fl=109-7 ° (1 A=0-1 nm). The unit cell volume is then 6"6 x 105 A 3, with probably a dimer as the asymmetric unit and two dimers per unit cell. This is probable since a calculation of the possible Vm value for the crystals gives 3"6 A a Da for two dimers and the unlikely value of 1"8 A a Da for four dimers per unit cell. On still photographs the diffraction pattern extends to 2 A.
References
Leberman, R., Berthat-Colominas, C., Cusack, S. & H~:rtlein, M. (1987). J. Mol. Biol. 193, 423-425. Saxton, W. 0., Pitt, T. J. & Homer, M. (1979). Ultramicroscopy, 4, 343-354. Yaremchuk, A. D., Tukalo, M. A., Konovalenko, A. V., Egorova, S. P. & Matsuka, G. Kh. (1989). Biopol. Kletka (U.S.S.R.), 5, 83-86.
Edited by A. Klug
Crystals of Seryl-tRNA Synthetase from Thermus thermophilus Preliminary Crystallographic Data M. B. Garber Institute of Protein Research, Academy of Sciences of the USSR 142292 Pushchino, Region, USSR
A. D. Yaremchuk, M. A. Tukalo, S. P. Egorova Institute of Molecular Biology and Genetics Academy oil Sciences of the Ukrainian SSR Kiev, USSR
C. Berthet-Colominas and R. Leberman E M B L Grenoble Outstation 156 X, 38042 Grenoble Cedex, France (Received 16 February 1990; accepted 6 March 1990) Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a=87"1 A, b= 126"9 A, c--63"5 A and fl= 109.7 °.
Crystallization trials were carried out using the hanging drop and vapour diffusion technique with 10-/~l drops of protein solution equilibrated against 500 #l of precipitant solution. Using ammonium sulphate solutions containing 5 mM-dithiothreitol 10 mM-MgCl2 1 mM-NaN3 (pH 6"2 to 8"0), very thin needle-like crystals were obtained. The morphology of these crystals was not improved by the addition of fl-octylglueoside. A variety of other additives were then tested, and it was found that the addition of methane pentane diol (MPDt) led to a marked improvement in crystal morphology. The final crystallization conditions were: drops containing 10 mg protein/ml and 20% saturated ammonium sulphate in 50 mM-Mes (pH 6"5), 5 mM-dithiothreitol, 10 mM-MgCt2, 1 mM-NaN3, 1% (v/v) MPD. These were equilibrated against 30 to 35% saturated ammonium sulphate containing 80/o MPD at room temperature, and crystals with dimensions of 0"3 mm x 0'5 m m x 1 mm could be obtained in a few days. These crystals, after washing with 50 % saturated ammonium sulphate solution containing 3%
Questions on the origin of thermostability of proteins might be answered by the study, at the atomic level, of the homologous protein from both mesophiles and thermophiles. Since in all organisms the aminoacyl-tRNA synthetases play an indispensable rSle in protein biosynthesis, the possibility of studying the same aminoacyl-tRNA synthetase from a mesophile and an extreme thermophile at the atomic level is an attractive proposition. The crystallization of seryl-tRNA synthetase from Escherichia coli has been described (Leberman et al., 1987) and a high-resolution structure of this enzyme is available (Cusack et al., unpublished results). We report the crystallization and preliminary crystallographic data of the homologous enzyme from the extreme thermophile Therrnus thermophilus. The purification of the seryl-tRNA synthetase (EC 6.1.1.11) from T. thermophilus has been described by Yaremchuk et al. (1989). The isolated enzyme is functional in aminoaeylating cognatetRNA from E. coll. Like the E. coli enzyme it is composed of two identical subunits but with a slightly smaller Mr value of about 46,000. The determination of the primary structure of the enzyme is currently underway. 0022-2836/90/120631-02 $03.00/0
Abbreviations used: MPD, methane pentane diol; Mes, 4-morpholino-ethanesulphonic acid. 631
~} 1990AcademicPress Limited
632
M . B . Garber et al.
MPD, were examined by SDS/polyacrylamide gel electrophoresis. A single band with a mobility corresponding to the original enzyme was found on Coomassie blue staining. The protein recovered from dissolved crystals also showed enzymic activity in the aminoacylation of tRNA. It should be noted that the enzyme is remarkably stable, retaining its integrity and biological activity after one year at room temperature under the crystallization conditions. A problem that arose was the handling of the crystals on mounting them in capillaries for X-ray diffraction studies in that using artificial mother liquor, they tended to dissolve. This problem was overcome by cross-linking with glutaraldehyde. Glutaraldehyde was added to the equilibration solution to a final concentration of 0"5% (v/v) once the crystals had grown, and they were left to equilibrate further for two days, then glutaraldehyde was added directly to the protein drop to a final concentration of 0"5~/o and left overnight. Crystals were then washed with a solution containing 30~o saturated ammonium sulphate and 2 ~/o MPD (pH 6"5). The cross-linked crystals were very stable and could be examined by X-ray diffraction. The crystals were mounted in glass capillaries and examined by CuKa X-radiation from a GX21 Elliot rotating anode generator using an Enraf-Nonius precession camera with a crystal-to-film distance of 60 mm. Unit cell dimensions were determined from l l ° precession photographs, by scanning the films with an Optronix rotating drum scanner (50 gm raster), followed by peak search and lattice refinement using subroutines of the SEMPER image-
processing package (Saxton et al., 1979). Crystals mounted with one edge parallel to the drum rotation axis gave precession photographs that showed cmm symmetry. This suggested that the apparent edge corresponds to a 2-fold axis; this was confirmed by the first upper level photograph that contained only the symmetry corresponding to the mirror perpendicular to the 2-fold axis, If the crystal was mounted with the same apparent edge parallel to the X-ray beam, then the precession photograph was of the hO1 plane. From precession photographs it was established that the crystals belong to the monoclinic space group P21, and that the unit cell parameters are a = 8 7 ' l A , b=126"9A, c = 6 3 . 5 A and fl=109-7 ° (1 A=0-1 nm). The unit cell volume is then 6"6 x 105 A 3, with probably a dimer as the asymmetric unit and two dimers per unit cell. This is probable since a calculation of the possible Vm value for the crystals gives 3"6 A a Da for two dimers and the unlikely value of 1"8 A a Da for four dimers per unit cell. On still photographs the diffraction pattern extends to 2 A.
References
Leberman, R., Berthat-Colominas, C., Cusack, S. & H~:rtlein, M. (1987). J. Mol. Biol. 193, 423-425. Saxton, W. 0., Pitt, T. J. & Homer, M. (1979). Ultramicroscopy, 4, 343-354. Yaremchuk, A. D., Tukalo, M. A., Konovalenko, A. V., Egorova, S. P. & Matsuka, G. Kh. (1989). Biopol. Kletka (U.S.S.R.), 5, 83-86.
Edited by A. Klug
