title: Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.
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.
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Cuttitta
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Ericson
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Scalia
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Roessler
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Ella
authors.initials: E
authors.lastname: Teplitsky
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Karan
authors.initials: K
authors.lastname: Joshi
authors.affiliation: Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Olven
authors.initials: O
authors.lastname: Campos
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Rakhi
authors.initials: R
authors.lastname: Agarwal
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Allaire
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Allen M
authors.initials: AM
authors.lastname: Orville
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Sweet
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Soares
mesh_terms: D000162:Acoustics; D003460:Crystallization; D057166:High-Throughput Screening Assays / Q000379:methods*; D020100:Hydrogels; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D020100:Hydrogels; D011506:Proteins; D012685:Sepharose
keywords: acoustic droplet ejection; chemical biology; crystal mounting; drug discovery; high-throughput screening; macromolecular crystallography