Optimization of crystallization conditions for biological macromolecules.

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters that influence crystallization, such as pH, ionic strength and precipitant concentration, as well as physical parameters such as temperature, sample volume and overall methodology. It also includes the application of some unique procedures and approaches, and the addition of novel components such as detergents, ligands or other small molecules that may enhance nucleation or crystal development. Here, an attempt is made to provide guidance on how optimization might best be applied to crystal-growth problems, and what parameters and factors might most profitably be explored to accelerate and achieve success.

Development of an alternative approach to protein crystallization.

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot.

High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 microm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 A structure demonstrating the feasibility of robotic protein crystal harvesting.

Searching for silver bullets: an alternative strategy for crystallizing macromolecules.

Based on a hypothesis that various small molecules might establish stabilizing, intermolecular, non covalent crosslinks in protein crystals and thereby promote lattice formation, we carried out three separate experiments. We assessed the impact of 200 chemicals on the propensity of 81 different proteins and viruses to crystallize. The experiments were comprised of 18240 vapor diffusion trials. A salient feature of the experiments was that, aside from the inclusion of the reagent mixes, only two fundamental crystallization conditions were used, 30% PEG 3350, and 50% Tacsimate at pH 7. Overall, 65 proteins (85%) were crystallized. Most significant was that 35 of the 65 (54%) crystallized only in the presence of one or more reagent mixes, but not in control samples lacking any additives. Among the most promising types of reagent mixes were those composed of polyvalent, charged groups, such as di and tri carboxylic acids, diamino compounds, molecules bearing one or more sulfonyl or phosphate groups, and a broad range of common biochemicals, coenzymes, biological effectors, and ligands. We propose that an alternate approach to crystallizing proteins might be developed, which employs a limited set of fundamental crystallization conditions combined with a broad screen of potentially useful small molecule additives.