Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

This article has been accepted and is currently in production

Abstract

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize a novel sample containing purified and concentrated protein, DNA, RNA, and their complexes, can however not be predicted. Subsequently, the yield of high quality crystals is usually very low. Since macromolecular crystallization is essentially a stochastic process with very little or no useful prior information, employing a broad variety of initial conditions should increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against more than 2,000 initial conditions by vapor diffusion in nanoliter droplets. Although our initial screen is often considered elsewhere as very large, it is still far from what would be an extensive screening of the suitable and useful combinations of crystallization reagents. Subsequently, the optimization of initial conditions is usually required. Semi-automated protocols have also been developed that facilitate the refinement of conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.