Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography

Abstract

Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation.

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

“…Recent developments enabled further miniaturization of experiments and hence the volume of sample required for screening crystallization conditions can be significantly reduced by integrating the corresponding technology 29 30 . However, some aspects of further miniaturization need careful consideration, such as the evaporation of droplets 31 and the manipulation of microcrystals 32 .…”

Using sound pulses to solve the crystal-harvesting bottleneck

“…After a crystal has been acoustically harvested, it can be rapidly combined with a chemical from (for example) a fragment library. The time and effort needed to harvest crystals for use in chemical library screening projects has driven efforts to use acoustic methods to improve the workflow for crystal growth (Wu et al, 2016), crystal harvesting (Chen et al, 2004) and chemical dispensation (Collins et al, 2017). Modified MiTeGen plates were used to explore simultaneous acceleration of crystal growth, crystal harvesting and chemical dispensation.…”

“…Acoustic droplet ejection (ADE) is an automated, keyboarddriven technology that can be used for growing protein crystals (Wu et al, 2016), improving the quality of protein crystals (Villaseñ or et al, 2010) and transferring protein crystals onto data-collection media (Soares et al, 2011) such as MiTeGen MicroMesh sample holders (hereafter referred to as 'micromeshes'). ADE can also be used to screen chemical libraries (Collins et al, 2017) using either cryocooled crystals (Yin et al, 2014) or room-temperature crystals (Teplitsky et al, 2015).…”

Gentle, fast and effective crystal soaking by acoustic dispensing

“…A technique that has been gaining utility for protein crystal applications is acoustic droplet ejection, a liquid-handling approach that relies on ultrasound pulses focused towards the surface of a liquid, thereby ejecting nanolitre or smaller volume droplets (Ellson et al, 2003). The precision and volume scales of acoustic transfer have enabled new developments in protein crystallography, including performing small-volume crystallization experiments in crystallization plates (Wu et al, 2016;Villaseñ or et al, 2012) or directly on data-collection mounts (Yin et al, 2014) and transferring preformed crystals into mounts (Cuttitta et al, 2015) or directly into the very short pulse of an XFEL beam (Roessler et al, 2016). Acoustic droplet ejection has also been used to prepare high-density crystallizations for in situ fragment screening (Teplitsky et al, 2015) where the precision and small volume handling of acoustic droplet ejection were used to fit 1728 crystallization experiments into a single microplate, and the same number of unique compounds could be added to existing crystals for soaking experiments, or added prior to crystallization and before drying as described above for dry cocrystallization.…”