The acceptor stem of Escherichia coli tRNAAla, rGGGGCUA[bull]rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3[bull]U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3′-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALAC70) determined at 1.4 Å exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3[bull]U70 wobble base pair may be functionally significant.