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Title: Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

Abstract

Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automatedmore » and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.« less

Authors:
 [1];  [2];  [3];  [4];  [5];  [6];  [7];  [8];  [9];  [10];  [7];  [7];  [11];  [7];  [7]
+ Show Author Affiliations
  1. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Stony Brook Univ., NY (United States). Dept. of Biochemistry and Cell Biology; Nanjing Univ. (China)
  2. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Binghamton Univ., NY (United States). Dept. of Biological Sciences
  3. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Ministry of Education of Brazil, Brasilia (Brazil). CAPES Foundation; Universidade Federal de Minas Gerais, Belo Horizonte-MG (Brazil)
  4. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; City Univ. of New York (CUNY), NY (United States). College of Staten Island. Dept. of Biology. Center for Developmental Neuroscience
  5. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; St Joseph’s College, East Patchogue, NY (United States)
  6. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Univ. at Buffalo, NY (United States). Dept. of Biomedical Engineering
  7. Brookhaven National Lab. (BNL), Upton, NY (United States). Photon Sciences Directorate
  8. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Florida Atlantic Univ., Boca Raton, FL (United States). Dept. of Biological Science
  9. Brookhaven National Lab. (BNL), Upton, NY (United States). Office of Education Programs; Comsewogue High School, Port Jefferson Station, NY (United States)
  10. Brookhaven National Lab. (BNL), Upton, NY (United States). Biosciences Dept.
  11. Brookhaven National Lab. (BNL), Upton, NY (United States). Photon Sciences Directorate; Brookhaven National Lab. (BNL), Upton, NY (United States). Biosciences Dept.
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division
OSTI Identifier:
1625713
Grant/Contract Number:  
SC0012704
Resource 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: 70; Journal Issue: 5; Journal ID: ISSN 1399-0047
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Biochemistry & Molecular Biology; Biophysics; Crystallography

Citation Formats

Yin, Xingyu, Scalia, Alexander, Leroy, Ludmila, Cuttitta, Christina M., Polizzo, Gina M., Ericson, Daniel L., Roessler, Christian G., Campos, Olven, Ma, Millie Y., Agarwal, Rakhi, Jackimowicz, Rick, Allaire, Marc, Orville, Allen M., Sweet, Robert M., and Soares, Alexei S. Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes. United States: N. p., 2014. Web. doi:10.1107/s1399004713034603.
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Yin, Xingyu, Scalia, Alexander, Leroy, Ludmila, Cuttitta, Christina M., Polizzo, Gina M., Ericson, Daniel L., Roessler, Christian G., Campos, Olven, Ma, Millie Y., Agarwal, Rakhi, Jackimowicz, Rick, Allaire, Marc, Orville, Allen M., Sweet, Robert M., & Soares, Alexei S. Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes. United States. https://doi.org/10.1107/s1399004713034603
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Yin, Xingyu, Scalia, Alexander, Leroy, Ludmila, Cuttitta, Christina M., Polizzo, Gina M., Ericson, Daniel L., Roessler, Christian G., Campos, Olven, Ma, Millie Y., Agarwal, Rakhi, Jackimowicz, Rick, Allaire, Marc, Orville, Allen M., Sweet, Robert M., and Soares, Alexei S. Wed . "Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes". United States. https://doi.org/10.1107/s1399004713034603. https://www.osti.gov/servlets/purl/1625713.
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@article{osti_1625713,
title = {Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes},
author = {Yin, Xingyu and Scalia, Alexander and Leroy, Ludmila and Cuttitta, Christina M. and Polizzo, Gina M. and Ericson, Daniel L. and Roessler, Christian G. and Campos, Olven and Ma, Millie Y. and Agarwal, Rakhi and Jackimowicz, Rick and Allaire, Marc and Orville, Allen M. and Sweet, Robert M. and Soares, Alexei S.},
abstractNote = {Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.},
doi = {10.1107/s1399004713034603},
journal = {Acta Crystallographica. Section D: Biological Crystallography (Online)},
number = 5,
volume = 70,
place = {United States},
year = {Wed Apr 30 00:00:00 EDT 2014},
month = {Wed Apr 30 00:00:00 EDT 2014}
}
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https://doi.org/10.1107/s1399004713034603
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