Fig. 7.
A. Electron density maps at 2.5 Å resolution, computed with amplitudes from cocrystals containing native, modified tRNA. The native data were first refined against the final model derived from the structure of GlnRS bound to the unmodified UUG anticodon tRNA and ATP. The gray map was then computed from amplitudes (2Fo-Fc) and is displayed at a contour level of 0.7 σ. The map shown in magenta was computed from amplitudes (Fo-Fc) and is displayed at a contour level of 3.0 σ. Peaks in the latter map adjacent to the exocyclic 2-oxygen and 5-carbon positions of U34 provide evidence for the presence of the modifications at these positions. The better-ordered surface loop spanning positions Lys444 to Gly449 appears in the (2Fo-Fc) map at bottom right. B. Interactions of cmnm5s2U34 with GlnRS. Left: primary structure of cmnm5s2U; Right: van der waals surfaces of atoms in the peptide spanning Arg410-Arg412 and at Lys444 are shown as dotted spheres, and hydrogen bonds are shown as dashed purple lines. The cmnm5 moiety lies adjacent to the backbone at Lys444 on the N-terminal end of the loop. The O2 moiety of unmodified U34 is displaced about 1.5 Å from the position occupied by the 2-thio group in cmnm5s2u34 (compare panel C). C. Superposition of the structures of GlnRS bound to the UUG anticodon transcript RNA (U34 shown in red), and bound to modified native tRNAGln containing cmnm5s2U34 (shown in blue). The loop spanning residues 444–454 is at bottom, with the salt bridge between Arg450 in the loop and adjacent Glu408 also depicted. The fully ordered loop in the native tRNA cocrystal is shown in green, and the partly disordered loop in the UUG structure is shown in yellow. The nearly identical orientations of the peptide containing Arg412 are shown at upper left. The relative orientation of the tRNA anticodon loop and protein backbone throughout the distal β-barrel domain is the same in these two structures. The side-chain of Arg410 is not shown for clarity (see panel B).