Nanolitre-scale crystallization using acoustic liquid-transfer technology

Abstract

Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.

Figure 1

Measurement of viscosity (green circles) and density (red squares) of solutions from the Classics (a) and JCSG+ (b) screens. The larger symbols represent solutions that failed to dispense and show no clear trend toward either extreme.

Figure 2

The distribution of coefficients of variation (CVs) for reproducibility in five crystallization screens calculated from six repeat dispenses. Each block of color shows the count of conditions from that screen within the prescribed range. The brown segment tallies wells that did not transfer and hence have no calculated CV.

Figure 3

Plots of the residual error in measured volume delivered relative to a 5 nl target for experiments at fixed energy throughout (light triangles) versus individually adjusted energies (dark circles). (a) Distribution of residuals across four screens (color-coded). (b) Enlarged view of conditions 13–28 of the PEG MW grid. Each set of four linked points is a progression of PEG concentration (5, 15, 20, 25%), illustrating the nature of the effect of the diminished measured volumes delivered as PEG increases (triangles). Application of a coarse energy correction (circles) demonstrates better delivery profiles (smaller range in residuals across PEG concentrations) and highlights further improvements to our energy-correction calculations to reduce overcompensation of the lowest measured residuals (e.g. points 20 and 24).

Figure 4

Images collected on day 15 from crystallization drops of various protein samples. The total drop volume is double the protein value given. The volumes are representative and were not optimized as similar crystals appeared at the various volumes explored. (a) HCV helicase (50 nl). (b) Human serum albumin (50 nl). (c) HCV polymerase (30 nl). (d) HIV RT (15 nl). (e) ITK (30 nl). (f) Lysozyme (15 nl).