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Agarose gel facilitates enzyme crystal soaking with a ligand analog
Author(s) -
Sauter Claude,
Balg Christian,
Moreno Abel,
Dhouib Kaouthar,
ThéobaldDietrich Anne,
Chênevert Robert,
Giegé Richard,
Lorber Bernard
Publication year - 2009
Publication title -
journal of applied crystallography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.429
H-Index - 162
ISSN - 1600-5767
DOI - 10.1107/s0021889809003446
Subject(s) - agarose , mosaicity , crystallization , orthorhombic crystal system , ligand (biochemistry) , chemistry , microcrystalline , amorphous solid , crystallography , molecule , crystal (programming language) , crystal structure , chemical engineering , chromatography , organic chemistry , biochemistry , receptor , epitaxy , layer (electronics) , computer science , programming language , engineering
Orthorhombic crystals of the enzyme aspartyl‐tRNA synthetase (AspRS) were prepared in agarose gel, a chemical alternative to microgravity or nano‐volume drops. Besides providing a convection‐free medium, the network of the polysaccharide improved the stability of the crystalline lattice during soaking with l ‐aspartol adenylate, a synthetic and non‐hydrolysable analog of the catalytic intermediate aspartyl adenylate. When crystals were embedded in the polysaccharide matrix the ligand reached their surfaces more uniformly. Gel‐grown crystals exhibited well defined reflections even at high resolution and low mosaicity values, despite their fairly high solvent content and the relatively harsh flash cooling procedure. By contrast, soaked AspRS crystals prepared in solution broke apart within 10–30 s after the ligand was introduced into the mother liquor, and subsequently these fragments became an amorphous precipitate. A general objection to the use of gels in protein crystallization is that chemical interactions may occur between the polysaccharide matrix and proteins or ligands. The example of AspRS shows that this is not a major concern. This method may be generally applicable to crystal soaking with other small molecules or heavy atoms.