A thermal stability assay can help to estimate the crystallization likelihood of biological samples
The identification of crystallization conditions for biological molecules largely relies on a trial‐and‐error process in which a number of parameters are explored in large screening experiments. Currently, construct design and sample formulation are recognized as critical variables in this process and often a number of protein variants are assayed for crystallization either sequentially or in parallel, which adds complexity to the screening process. Significant effort is dedicated to sample characterization and quality‐control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. However, large‐scale studies relating crystallization success to sample properties are generally lacking. In this study, the thermal stability of 657 samples was estimated using a simplified Thermofluor assay. These samples were also subjected to automated vapour‐diffusion crystallization screening under a constant protocol. Analysis of the data shows that samples with an apparent melting temperature (T m) of 318 K or higher crystallized in 49% of cases, while the crystallization success rate decreased rapidly for samples with lower T m. Only 23% of samples with a T m below 316 K produced crystals. Based on this analysis, a simple method for estimation of the crystallization likelihood of biological samples is proposed. This method is easy, rapid and consumes very small amounts of sample. The results of this assay can be used to determine optimal incubation temperatures for crystallization experiments or to prioritize certain constructs. More generally, this work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects.
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Document Type: Research Article
Publication date: 2011-11-01