Native mrna 5 aaauuu3

We purified and crystallized 30S ribosomal subunits from T. thermophilus HB8 strain essentially as described^{27 (link),28 (link)}. The m6A modified mRNA fragments (with codon sequences underlined), 5′(m6A)AAUUU3′, 5′A(m6A)AUUU3′, 5′AA(m6A)UUU3′ and native mRNA 5′AAAUUU3′ were purchased from Dharmacon. The presence of m6A modification was confirmed by mass spectrometry. The ASL
$\begin{array}{c}\mathit{\text{Lys}}3\\ \mathit{\text{UUU}}\end{array}$ , (with anticodon sequence underlined GCAGACU(_mcm5s2U)UU(_ms2t6A)AΨCUGC), is a generous gift of Paul Agris (University of Albany). 30S crystals were sequentially transferred to the final buffer with 26% v/v 2-methyl-2,4-pentanediol (MPD) for cryoprotection and soaks were performed in the final buffer supplemented with 200μM of each mRNA oligos, ASL
$\begin{array}{c}\mathit{\text{Lys}}3\\ \mathit{\text{UUU}}\end{array}$ and 80μM paromomycin for 48hours. Crystals were flash-frozen for data collection by plunging them directly into liquid nitrogen.