Advanced Biocrystallogenesis

Nowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical parameters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.

A beginner’s guide to macromolecular crystallization

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

An anticipated optimization approach to macromolecular crystallization

Crystallization is an essential step for determining macromolecular structures at atomic resolution with X-ray crystallography. Crystals of diffraction quality obtained from purified samples of proteins, RNAs, DNAs and their complexes enable our understanding of biological processes and structure-based design of drugs. Targets of interest for researchers are however increasingly challenging to produce and crystallise. Progress in crystallization methods applicable to limiting amount of sample while increasing the yield of useful crystals are hence urgently needed. In this context, an Anticipated Optimization Approach was investigated. For this approach, it is assumed that samples are highly unstable and will most probably not produce useful crystals (‘hits’). By selecting leads very early, what remains from the initial sample can be used for follow-up optimization experiments. Subsequently, the reproducibility issues caused by sample variability are bypassed. An initial crystallization screen that failed to produce hits becomes a well-suited solubility assay that is a starting point for optimization. The approach was tested with a straightforward and cost-effective protocol developed elsewhere. The yield of useful crystals was increased and accelerated for three targets of pharmacologic studies.SynopsisThis study suggests that the yield of useful crystals obtained from challenging protein samples can be increased by using an initial crystallization screen as solubility assay.

Iterative screen optimization maximizes the efficiency of macromolecular crystallization

Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations.

Berkeley Screen: a set of 96 solutions for general macromolecular crystallization

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography program at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens.