Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P.
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX-LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a=54.80, b=69.15, c=94.08 A, alpha=95.47, beta=106.51, gamma=90.46 degrees, and diffracted to 1.9 A resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX-EF-P-LysAMS crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=105.93, b=102.96, c=119.94 A, beta=99.4 degrees, and diffracted to 2.5 A resolution. Structure determination of the E. coli GenX-LysAMS and GenX-EF-P-LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.
Sumida
Tomomi
T
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.
Yanagisawa
Tatsuo
T
Ishii
Ryohei
R
Yokoyama
Shigeyuki
S
D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs
D010445:Peptide Elongation Factors; C020746:factor EF-P; D008250:Lysine-tRNA Ligase
Trends Biochem Sci. 2000 May;25(5):207-9
10782085
Biol Direct. 2010 Jan 13;5:3
20070887
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Methods Enzymol. 1997;276:307-26
27754618
Annu Rev Biochem. 1987;56:125-58
3304131
Microbiol Mol Biol Rev. 2002 Sep;66(3):460-85, table of contents
12209000
EMBO J. 1996 Nov 15;15(22):6321-34
8947055
Nature. 1990 Sep 13;347(6289):203-6
2203971
Biochemistry. 2000 Jul 25;39(29):8418-25
10913247
Gene. 1991 Dec 1;108(1):163-4
1761227
Structure. 1995 Feb 15;3(2):163-76
7735833
Eur J Biochem. 1979 Jun;97(1):23-8
383483
Nucleic Acids Res. 2005 Jan 1;33(Database issue):D433-7
15608232
FEBS Lett. 2010 Jan 21;584(2):460-6
19913539
Science. 2009 Aug 21;325(5943):966-70
19696344
Infect Immun. 1998 Dec;66(12):5599-606
9826331
J Bacteriol. 2001 Jan;183(1):36-45
11114898
Proc Natl Acad Sci U S A. 1975 Nov;72(11):4257-60
1105576
Nature. 1990 Sep 20;347(6290):249-55
2205803
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9595-600
15210970