| Autor: |
Yin X; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Scalia A; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Leroy L; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Cuttitta CM; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Polizzo GM; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Ericson DL; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Roessler CG; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Campos O; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Ma MY; Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Agarwal R; Biosciences Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Jackimowicz R; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Allaire M; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Orville AM; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Sweet RM; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA., Soares AS; Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973-5000, USA. |
| Abstrakt: |
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. |
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