Computational protocol: B12 cofactors directly stabilize an mRNA regulatory switch

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Protocol publication

[…] A representative sequence from the original sequence alignment (Genbank Accession #AACY021350931.1/557-442) was selected. The P13 stem was removed and the anti-RBS sequence in P5 was replaced with a stable GAAA tetraloop. The RNA was synthesized and refolded as described above. Diffraction quality crystals grew within 3 days at 30 °C in the presence of 100 mM magnesium acetate, 10% 2-methyl-1,3 propanediol (MPD), and 5 mM iridium hexammine. Crystals were cryoprotected in Ficoll oil and flash frozen in liquid nitrogen. Complete datasets at the iridium and cobalt anomalous edges were collected and processed via autoPROC. The structure was solved using PHENIX. The density modified map showed excellent density for the entire RNA (). The model was built manually in COOT and refined with BUSTER. […]

Pipeline specifications

Software tools PHENIX, Coot
Application Protein structure analysis
Chemicals Hydrogen, Vitamin B 12