The development of a robust industrial crystallography program for the bacterial 50S ribosomal subunit

Although structure-based drug design (SBDD) is now a core technology in drug development, access to SBDD of the ribosome can still be limited by difficulties in obtaining high quality crystals. We have systematically assessed correlations between crystallization and the purity, integrity and activity of 50S ribosomal subunits from Deinococcus radiodurans as part of a program to develop a robust SBDD program. The work has informed changes to purification that have increased the success rate so that every preparation crystallizes without fail. Crystals diffract similarly to those from other laboratories, but with routine generation of crystals large enough to obtain a whole data set from a single crystal. The effects of altering purification parameters on subsequent crystallization are reviewed, including two novel ribosome chromatography methods that we have developed as alternatives to standard purification by centrifugation. The resulting system now satisfies our needs to inform synthesis of antibiotic candidates, with a one month turnaround between selecting compounds and delivering co-crystal structures.