Abstract: We have re-determined the crystal structure of yeast tRNA(Phe) to 2. 0 A resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg(2+) is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg(2+ )binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TPsiC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg(2+).CI - Copyright 2000 Academic Press.