A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

Abstract

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.

MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines

“…However, the results presented here show that this clearly does not need to be the case and that complete, highquality data sets could straightforwardly be compiled from a series of smaller crystals mounted on the same sample holder. Moreover, as evidence suggests that smaller crystals require reduced fragment/ligand-soaking times to obtain the same occupancy of the fragment/ligand in crystal structures (Cole et al, 2014), microcrystal-based fragment screening experiments may well become the norm, with soaking times based on the largest crystal contained in the crystallization drop ensuring the maximum occupancy of ligands/fragments in all of the crystals mounted on a single sample loop. research papers 4.2.2.…”

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

“…While capillary and microfluidic devices can clog and damage crystals by subjecting them to shear force, in ADE the droplet emerges from the meniscus of the fluid without passing through an orifice. Some current applications of ADE in macromolecular crystallography (MX) include crystal growth (Villaseñor et al, 2012), microseeding (Villaseñor et al, 2010), ligand screening (Cole et al, 2014; Teplitsky et al, 2015; Yin et al, 2014), and discrete crystal deposition onto data collection media for synchrotron-based cryo-crystallography (Cuttitta et al, 2015; Heroux et al, 2014; Roessler et al, 2013; Soares et al, 2011). …”

Using sound pulses to solve the crystal-harvesting bottleneck

“…The most important improvement required is a crystallization plate that is designed for acoustic compatibility. High-throughput screening applications are a natural first fit for acoustic harvesting; small crystals are particularly suitable because they are easy to eject and because they combine rapidly with chemical libraries (Cole et al, 2014). Our experience is that cuboidal crystals larger than 50 mm occasionally fail to eject, and crystals larger than 160 mm rarely eject (although much larger rod-shaped crystals can be ejected).…”