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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.

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Inhibitor soaks of cdk2 gave crystals that diffracted to ∼2 Å. Dilute inhibitor concentrations coupled with long incubation times gave the best complexes.
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fig19: Inhibitor soaks of cdk2 gave crystals that diffracted to ∼2 Å. Dilute inhibitor concentrations coupled with long incubation times gave the best complexes.

Mentions: The previous example showed crystals of cyclinA–cdk2 that were sensitive to handling where cross-linking and the use of an additive were critical for successful ligand soaks. However, in cdk2, a second approach of diluting the inhibitor and using longer incubation times worked well in obtaining inhibitor complexes. 1–3 µl of a 50 mM stock inhibitor solution was added to 500 µl well reservoir. 1 µl of this diluted ligand was then added to a 4–5 µl drop containing the cdk2 crystals (Fig. 19 ▶). Incubation times ranged from several days to 2–3 months.


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)

Inhibitor soaks of cdk2 gave crystals that diffracted to ∼2 Å. Dilute inhibitor concentrations coupled with long incubation times gave the best complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig19: Inhibitor soaks of cdk2 gave crystals that diffracted to ∼2 Å. Dilute inhibitor concentrations coupled with long incubation times gave the best complexes.
Mentions: The previous example showed crystals of cyclinA–cdk2 that were sensitive to handling where cross-linking and the use of an additive were critical for successful ligand soaks. However, in cdk2, a second approach of diluting the inhibitor and using longer incubation times worked well in obtaining inhibitor complexes. 1–3 µl of a 50 mM stock inhibitor solution was added to 500 µl well reservoir. 1 µl of this diluted ligand was then added to a 4–5 µl drop containing the cdk2 crystals (Fig. 19 ▶). Incubation times ranged from several days to 2–3 months.

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