Limits...
Crystallization of protein-ligand complexes.

Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule.These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form.This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Computational, Analytical and Structural Sciences, Glaxo SmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA. annie.m.hassell@gsk.com

ABSTRACT
Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Show MeSH
Protease 1. The use of xylitol during the soaking stabilized the crystal and improved the solubility of the ligands. If the ligand was not mixed in the xylitol plus precipitating mixture, the efficiency of obtaining protein–ligand complexes greatly decreased.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483499&req=5

fig18: Protease 1. The use of xylitol during the soaking stabilized the crystal and improved the solubility of the ligands. If the ligand was not mixed in the xylitol plus precipitating mixture, the efficiency of obtaining protein–ligand complexes greatly decreased.

Mentions: In protease 1, cocrystallizations yielded very few protein–ligand complexes, so a soaking strategy was devised. Since the apo crystals were rather fragile and the ligands were quite insoluble, an additive was needed that stabilized the crystal and improved the ligand solubility. Xylitol was added to the precipitating solution to a final concentration of ∼2–5%. 1 µl of this mixture was added to the protein drop to stabilize the crystals (∼15–60 min). Next, the inhibitor was added to the additive plus precipitating reagent mix (∼2–5 µl ligand plus 500 µl precipitating reagent). 1 µl of this ligand mixture was then added to the crystals (Fig. 18 ▶). This procedure was the only method that led to solution of structures of the protease–ligand complexes.


Crystallization of protein-ligand complexes.

Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM - Acta Crystallogr. D Biol. Crystallogr. (2006)

Protease 1. The use of xylitol during the soaking stabilized the crystal and improved the solubility of the ligands. If the ligand was not mixed in the xylitol plus precipitating mixture, the efficiency of obtaining protein–ligand complexes greatly decreased.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483499&req=5

fig18: Protease 1. The use of xylitol during the soaking stabilized the crystal and improved the solubility of the ligands. If the ligand was not mixed in the xylitol plus precipitating mixture, the efficiency of obtaining protein–ligand complexes greatly decreased.
Mentions: In protease 1, cocrystallizations yielded very few protein–ligand complexes, so a soaking strategy was devised. Since the apo crystals were rather fragile and the ligands were quite insoluble, an additive was needed that stabilized the crystal and improved the ligand solubility. Xylitol was added to the precipitating solution to a final concentration of ∼2–5%. 1 µl of this mixture was added to the protein drop to stabilize the crystals (∼15–60 min). Next, the inhibitor was added to the additive plus precipitating reagent mix (∼2–5 µl ligand plus 500 µl precipitating reagent). 1 µl of this ligand mixture was then added to the crystals (Fig. 18 ▶). This procedure was the only method that led to solution of structures of the protease–ligand complexes.

Bottom Line: Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule.These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form.This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Computational, Analytical and Structural Sciences, Glaxo SmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA. annie.m.hassell@gsk.com

ABSTRACT
Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Show MeSH