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Title
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Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes.
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Authors
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X.Yin,
A.Scalia,
L.Leroy,
C.M.Cuttitta,
G.M.Polizzo,
D.L.Ericson,
C.G.Roessler,
O.Campos,
M.Y.Ma,
R.Agarwal,
R.Jackimowicz,
M.Allaire,
A.M.Orville,
R.M.Sweet,
A.S.Soares.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2014,
70,
1177-1189.
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PubMed id
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Abstract
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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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