Crystallization of RNAProtein Complexes: From Synthesis and Purification of Individual Components to Crystals

A broad range of biological processes relies on complexes between RNA and proteins. Crystallization of RNA–protein complexes can yield invaluable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA–protein complexes is often challenging and requires special approaches. Here we review the purification of RNA, RNA-binding proteins, and the formation and crystallization of RNA–protein complexes, using the crystallization of the P3 RNA domain of ribonuclease MRP, a multicomponent ribonucleoprotein complex involved in the metabolism of various RNA molecules, as an example. The RNA–protein complex was formed using gel-purified RNA, produced by run-off transcription with T7 RNA polymerase in vitro, and proteins that were overexpressed in Escherichia coli and purified to be RNase-free. The complex was crystallized using a sitting drop setup; initial screening for suitable crystallization conditions was performed using a sparse matrix approach.