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

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 proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to 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 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize 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.