Isolation, crystallization and X-ray analysis of the quaternary complex of Phe-tRNAPhe, EF-Tu, a GTP analog and kirromycin

AbstractKirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Complexes of the antibiotic, Phe-tRNAPhe, the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF-Tu were isolated. Crystallization was achieved at 4°C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 Å, and angles of 54.0°. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe-tRNAPhe, GDPNP, and Thermus aquaticus EF-Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464–1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 Å. In both cases, the crystals contain one complex per asymmetric unit