A Straightforward and Robust Method for Introducing Human Hair as a Nucleant into High Throughput Crystallization Trials

Abstract

Growing X-ray grade crystals of a specific protein is a process of trial and error. Usually, hundreds or even thousands of conditions are screened in order to identify useful crystallization conditions. Heterogeneous nucleants have been shown to increase the success rate of crystallization trials, and (human) hair has previously been identified as a promising nucleant. Here, we describe and evaluate a method for preparing crystallization plates that are locally coated with fragments of human hair, allowing automated, high-throughput crystallization trials in a fashion that is entirely compatible with standard hanging or sitting drop crystallization techniques. We assessed the effect of these nucleants on the crystallization of 11 different proteins in more than 4000 crystallization trials. We found additional crystallization conditions for 10 out of 11 proteins when using the standard JCSG+ screen (96 different conditions). In total, 34 additional crystallization conditions could be identified (13.1% of the total number of successful crystallizations). The increase in crystallization conditions ranged between 33.3% (two additional conditions were identified for myoglobin on top of four homogeneous crystallizations) to 1.2% (we identified a single additional condition for insulin, which crystallized in 85 out of 96 conditions); the median increase in crystallization hits was 14%. On the basis of these numbers, we conclude that the inclusion of human hair fragments in high throughput crystallization screens may be beneficial. The method is inexpensive, straightforward with standard equipment and uses materials available in any crystallization lab. Furthermore, initial experiments with the crystallization of membrane proteins on hair show the technique may also be beneficial for growing membrane proteins.