Determination and uses of the atomic structures of ribosomes and ribosomal subunits and their ligand complexes
The present invention provides methods for producing high resolution crystals of ribosomes and ribosomal subunits as well as the crystals produced by such methods. The x-ray diffraction patterns of the crystals provided by the present invention are of sufficiently high resolution for determining the three-dimensional structure of ribosomes and ribosomal subunits, for identifying ligand binding sites on ribosomes and ribosomal subunits, and for molecular modeling of ligands which interact with ribosomes and ribosomal subunits. The present invention provides methods for identifying ribosome-related ligands and methods for designing ligands with specific ribosome-binding properties. Thus, the methods of the present invention may be used to produce ligands which are designed to kill or inhibit any specific target organism(s).
1. A method of growing a crystal of a 50S ribosomal subunit from Haloarcula marismortui comprising:
(a) isolating a 50S ribosomal subunit from Haloarcula marismortui ;
(b) precipitating the 50S ribosomal subunit;
(c) back-extracting or resuspending the precipitated 50S ribosomal subunit to obtain a solution;
(d) seeding the back-extracted or resuspended solution of step (c);
(e) growing a crystal of the 50S ribosomal subunit from the seeded solution of step (d) by vapor diffusion at room temperature;
(f) harvesting the crystal from step (e);
(g) stabilizing the harvested crystal by gradual transfer of said crystal into a series of solution containing high salt concentration of from about 1.2 M to 1.7 M; and
(h) maintaining the crystal under high salt concentration, wherein the crystal (i) is untwinned, (ii) has an average thickness greater than about 15 μm, and (iii) diffracts X-rays to a resolution of at least 2.7 Å.
2. The method of claim 1 further comprising:
(i) flash freezing the crystal of step (h).