High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

Abstract

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Figure 1:. Schematic of a high-throughput 1,536-well crystallization screening experiment performed at the HTX Center.

(1) In this step, 5 μL of paraffin oil and 200 nL of cocktail are added to each well (protocol step 3.1 and step 3.5). A cartoon illustration of one well containing only oil and cocktail and a representative image are shown to the right. (2) Samples arrive at the HTX Center (protocol step 5.1). 3) In this step, 200 nL of sample is added to each well (protocol step 5.4). (4) All 1,536 wells are monitored over time using brightfield imaging, 5) as well as the UV-TPEF and SHG modalities (protocol step 6). 6) The AI-enabled open-source GUI is used to view, score, and analyze the crystallization images (protocol step 7). Abbreviations: HTX = high-throughput crystallization; UV-TPEF = UV-two-photon excited fluorescence; SHG = second harmonic generation; AI = artificial intelligence; GUI = graphical user interface.

Figure 2:. Single 1,536-well plates containing screening experiments, imaged using brightfield, UV-TPEF, and SHG imaging.

The 1,536-well plates are shown with an American penny for scale (top). Each screening experiment is imaged once prior to setup and six times after sample addition with brightfield imaging (seven total brightfield image sets, left). The plates undergo UV-TPEF (center) and SHG (right) imaging at 4 weeks or 6 weeks. Abbreviations: UV-TPEF = UV-two-photon excited fluorescence; SHG = second harmonic generation.

Figure 3:. Schematic showing how the 1,536-well plates are generated.

Sixteen 96-well DW blocks are used to stamp out four 384-well plates, with each quadrant of each 384-well plate filled by dispensing crystallization cocktails. Four 96-well DW blocks fill one 384-well plate (middle). Four 384-well plates are used to stamp out the single 1,536-well plate (right). Abbreviation: DW = deep well.

Figure 4:. Representative time course of a single well in a 1,536-well screening experiment.

Plates are imaged prior to sample setup (day 0), as well as with brightfield imaging on day 1, week 1, week 2, week 3, week 4, and week 6. The plates incubated at 23 °C are imaged with SONICC at week 4. Scale bars = 80 μm (brightfield), 200 μm (SHG, UV-TPEF). Abbreviations: SONICC = second order nonlinear imaging of chiral crystals; UV-TPEF = UV-two-photon excited fluorescence; SHG = second harmonic generation.

Figure 5:. Representative imaging results for the HT 1,536 crystal screening experiments.

Brightfield, UV-TPEF, and SHG imaging results are shown for five example wells. (A,B) Protein crystals observed by brightfield, UV-TPEF, and SHG imaging are clearly apparent in all three imaging modalities. (C) A protein crystal obscured by film in brightfield imaging is visible by UV-TPEF imaging; the crystal is not observed by SHG imaging due to point group incompatibility. (D) Example of microcrystals verified by UV-TPEF and SHG imaging that may otherwise be considered precipitate. (E) Example of salt crystals that appear crystalline by brightfield and SHG imaging but do not exhibit a UV-TPEF signal. Scale bars = 200 μm. Well diameter = 0.9 mm. Abbreviations: UV-TPEF = UV-two-photon excited fluorescence; SHG = second harmonic generation.