Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

Christina M Cuttitta,Christian G Roessler,Alexander Scalia,Ella Teplitsky,Allen M Orville,Olven Campos,Karan Joshi,Rakhi Agarwal,Robert M Sweet,Marc Allaire,Alexei S Soares,Daniel L Ericson

doi:10.1107/s1399004714013728

Abstract

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

Highlights

Acoustic droplet ejection (ADE) is an automated and keyboard-driven technology for growing protein crystals (Yin et al, 2014; Villasenor et al, 2012), improving the quality of protein crystals (Villasenor et al, 2010) and transferring protein crystals onto data-collection media (Soares et al, 2011) such as pin-mounted micromesh sample holders
Any solution that appeared to form a precipitate was recorded (CAPs were examined with a light microscope and any discoloration was noted as a precipitate)
This study reports the use of agarose gels to support protein crystals at a suitable location for automatic crystal transfer using ADE

Summary

Introduction

Acoustic droplet ejection (ADE) is an automated and keyboard-driven technology for growing protein crystals (Yin et al, 2014; Villasenor et al, 2012), improving the quality of protein crystals (Villasenor et al, 2010) and transferring protein crystals onto data-collection media (Soares et al, 2011) such as pin-mounted micromesh sample holders. High-throughput screening of chemical libraries (such as fragment libraries) using X-ray crystallography requires a fast and flexible crystal-mounting technology. Acoustic crystal mounting is an attractive choice for high-throughput screening applications (Table 1). Since ADE is automated, its success is not dependent on the manual dexterity or physical aptitude of. ADE is gentle in that no tools (for example pipette tips) touch

Methods

Results

Conclusion