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

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, whichmore » 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.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ;  [6] ;  [7] ;  [8] ;  [4] ;  [4] ;  [4] ;  [4]
  1. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; City Univ. of New York, Staten Island, NY (United States); Center for Developmental Neuroscience and Dept. of Biology
  2. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; Univ. at Buffalo, SUNY, NY (United States). Dept. of Biomedical Engineering
  3. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; Binghamton Univ., NY (United States). Dept. of Biological Sciences
  4. Brookhaven National Lab. (BNL), Upton, NY (United States). Photon Sciences Directorate
  5. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; Stony Brook Univ., NY (United States). Dept. of Biochemistry and Cell Biology
  6. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; PEC Univ. of Technology, Chandigarh (India). Dept. of Electronics and Electrical Communication Engineering
  7. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Educational Programs; Florida Atlantic Univ., Boca Raton, FL (United States). Dept. of Biological Science
  8. Brookhaven National Lab. (BNL), Upton, NY (United States). Biosciences Dept.
Publication Date:
OSTI Identifier:
1210164
Report Number(s):
BNL--107952-2015-JA
Journal ID: ISSN 1399-0047; ABCRE6; R&D Project: LS001
Grant/Contract Number:
SC00112704
Type:
Accepted Manuscript
Journal Name:
Acta Crystallographica. Section D: Biological Crystallography (Online)
Additional Journal Information:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography (Online); Journal Volume: 71; Journal Issue: 1; Journal ID: ISSN 1399-0047
Publisher:
International Union of Crystallography
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE