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

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2014-05, Vol.70 (Pt 5)
• Main Authors: Yin, Xingyu, Stony Brook University, NY 11794-5215, Nanjing University, Nanjing, Jiangsu, Scalia, Alexander, Binghamton University, 4400 Vestal Parkway East, NY 13902, Leroy, Ludmila, Ministry of Education of Brazil, 70040-020 Brasilia-DF, Universidade Federal de Minas Gerais, 6627 Av. Antonio Carlos, 31270-901 Belo Horizonte-MG, Cuttitta, Christina M., The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, Polizzo, Gina M., St Joseph’s College, 155 West Roe Boulevard, East Patchogue, NY 11772, Ericson, Daniel L., University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260, Roessler, Christian G., Campos, Olven, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414, Ma, Millie Y., Comsewogue High School, 565 Bicycle Path, Port Jefferson Station, NY 11776, Agarwal, Rakhi, Jackimowicz, Rick, Allaire, Marc, Orville, Allen M., Brookhaven National Laboratory, Upton, NY 11973-5000, Sweet, Robert M., Soares, Alexei S.
• Format: Article
• Language: English
• Subjects: ACCURACY; CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALLOGRAPHY; CRYSTALS; DROPLETS; ENVIRONMENT; EQUILIBRIUM; IRON; LIQUIDS; LYSOZYME; SCREENING; SYNCHROTRON RADIATION; TRAJECTORIES; VELOCITY
• ISSN: 0907-4449; 1399-0047

A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin. 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.