The importance of developing miniaturized and automated assays for the crystallization of biological macromolecules

Our blog

19 February, 2014

Fabrice Gorrec, MRC-LMB, Cambridge, UK

X-ray crystallography still plays a dominant role in solving biomolecular structures. Collaborations with structural biologists are encouraged now that only small amounts of protein are required and the process of structure determination is efficient enough for the majority of biologists and biochemists to employ it. Numerous innovations and technological developments have assisted progress in BioMolecular Crystallography (BMC), notably in the fields of molecular biology, IT and synchrotron light sources.

BMC will ultimately always depend on the production of quality crystals. Crystallization had become the bottleneck of the structure determination process due to the increasingly challenging nature of novel biological samples of interest. When X-ray structure determination evolved into a standard technique for researchers to apply to their favored proteins, crystallization became a problem and has now evolved into a science in itself. Nevertheless, the approach is still empirical: at the time of the crystallization experiments, nothing is known about the biomacromolecule to be assembled into an ordered periodic crystal lattice. The challenge for crystallographers is how to solve a puzzle without knowing the shape of the main pieces.

Beyond crystallization, one has to look at the BMC process overall, with its multitude of steps, each one of which is potentially a dead-end. The worst is late stage failure (failure to analyze diffraction data because of “the phase problem” is frequent). A parallel approach is strongly recommended, with different trials at each stage of the process in anticipation of the difficulties that one could confront in the next stage. In addition to multiple sample variants, one will employ a variety of crystallization screens.

There are an almost infinite number of parameter combinations when attempting to solve a structure. Most of these parameters alter the availability and quality of the required crystals. A challenging crystal structure generally results from thousands – or hundreds of thousands – of crystallization assays in initial screening and optimization experiments. This volume has driven developments in automation and miniaturization.

Miniaturization enables the use of a multitude of crystallization conditions even when the amount of sample is limited. Automation enables one to proceed quickly, accurately and effectively. Speed is important because samples typically have poor stability. Ensuring accuracy and repeatability reduces eventual reproducibility issues to a minimum (optimizing with a subsequent batch of protein or a sample that is not freshly prepared frequently results in failure). Finally, cost-effectiveness is particularly relevant for difficult, long-term, projects. It is hard to imagine the process of structure determination can be fully automated soon, because of its complexity and the level of know-how required for each step. Nevertheless, hands-free macromolecular crystallization in nanolitre drops has already been successfully enabled with commercially available robots (Figure 1).




Figure 1. Fully automated system for high-throughput crystallization in nanolitre drops. The plates containing the crystallization conditions are laid on the deck of a liquid handling robot with a customized aluminum lid on top. A gripper removes the lid and places a plate on the deck of the mosquito® Crystal robot for setting up nanolitre drops. The plate is later transported to a sealer (for sealing with transparent tape). Finally, the plate is placed back onto its original location (crystallization experiments start).





Fabrice Gorrec gained experience in Structural Biology and Drug Discovery at GlaxoSmithKline (2003-4) and later at the Structural Genomics Consortium (2005-6). Since 2007, Fabrice has been responsible for the Macromolecular Crystallization Robotic Facility at the Medical Research Council – Laboratory of Molecular Biology (MRC-LMB, Cambridge, UK). He works with research scientists to enable extensive initial crystallization screening and later crystal optimization, always lending his support to tackling difficult, long-term research problems. In 2012, Fabrice received an MRC Award for innovations in the field of macromolecular crystallization.