Abstract
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.
Highlights
Macromolecular X-ray crystallography has undergone dramatic advances since the crystal structure of myoglobin was first reported (Kendrew et al, 1958)
Many drops that were heavily precipitated in Plate 1 were no longer scored as heavily precipitated after their precipitant concentration was decreased by 20% in the first round of optimization
The Sweet16 crystallization screen that we designed proved to be an effective platform for iterative screen optimization (ISO), this method can be applied to any crystallographic screening strategy, regardless of the conditions that make up the initial screen
Summary
Macromolecular X-ray crystallography has undergone dramatic advances since the crystal structure of myoglobin was first reported (Kendrew et al, 1958). Diffraction data can be rapidly processed and structures determined with the use of software packages such as CCP4, PHENIX and Coot (Winn et al, 2011; Adams et al, 2002; Emsley et al, 2010) Thanks to these technological advances, it has become possible for crystallographers to generate high-resolution molecular models in days or weeks under ideal conditions. Both of these methods have been heavily utilized for crystallographic screening, but each suffers from disadvantages. Free-interface diffusion more thoroughly probes the crystallographic phase diagram for a given set of chemical conditions; its unique experimental platform is costly and makes it difficult to isolate crystals for subsequent diffraction experiments
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