Crystallographic analysis of the Escherichia coli tRNA seleno-modification enzyme in complex with tRNA

The bacterial enzyme tRNA 2-selenouridine synthase (SelU) catalyzes the conversion of 5-substituted 2-thiouridine (R5S2U) to 5-substituted 2-selenouridine (R5Se2U) at the wobble positions of several tRNAs. Seleno-modification potentially regulates translation efficiency in response to selenium availability. Notably, SelU uses the 2-geranylthiouridine (R5geS2U) intermediate for sulfur removal, and this geranylthiol (geS) is a unique leaving group among tRNA-maturation enzymes. However, the underlying sequence of the SelU reaction remains unclear. Here, a crystallographic study of the Escherichia coli SelU–tRNA complex is reported. Robust and well formed SelU–tRNA crystals were obtained after several optimizations, including co-expression with tRNA and additive screening. Diffraction data were collected at a resolution of 3.10 Å using a wavelength of 1.0000 Å. The crystals belonged to space group C2, and the phase was determined by molecular replacement using the AlphaFold2-predicted SelU structure as a search model. Electron-density mapping revealed the presence of two SelU–tRNA complexes in the asymmetric unit.