RT Journal Article SR Electronic T1 Gentle, fast and effective crystal soaking by acoustic dispensing JF bioRxiv FD Cold Spring Harbor Laboratory SP 085712 DO 10.1101/085712 A1 Patrick M Collins A1 Jia Tsing Ng A1 Romain Talon A1 Karolina Nekrosiute A1 Tobias Krojer A1 Alice Douangamath A1 Jose Brandao-Neto A1 Nathan Wright A1 Nicholas M Pearce A1 Frank von Delft YR 2016 UL http://biorxiv.org/content/early/2016/11/04/085712.abstract AB Synopsis A high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness demonstrated. Abstract Bright light sources, agile robotics, and fast detectors are continually reducing the time it takes to perform an X-ray diffraction experiment, making high throughput experiments more feasible than ever. But this is also pushing the upstream bottleneck towards sample preparation, even for robust and well characterised crystal systems. Crystal soaking is routinely used to generate protein-ligand complex structures, yet protein crystals are often sensitive to changes in solvent composition, and frequently require gentle or careful stepwise soaking techniques, limiting overall throughput. Here, we describe the use of acoustic droplet ejection for soaking of protein crystals with small molecules, and show that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 minutes. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables targeted placement of the compound/solvent away from crystals and towards drop edges, allowing for gradual diffusion of solvent across the drop. This ensures both an improvement in reproducibility of X-ray diffraction and an increased solvent tolerance of the crystals, thus enabling higher effective compound soaking concentrations. We detail the technique here with examples from the protein target JMJD2D, a histone lysine demethylase, having roles in cancer and the focus of active structure based drug design efforts.