title: Preparation, Crystallization, and Structure Determination of Chromatin Enzyme/Nucleosome Complexes.
abstract: Structural studies of chromatin complexes composed of chromatin factors or enzymes bound to the nucleosome have been constrained by the ability to produce high-quality complexes in the amounts appropriate for biophysical studies and by the difficulty of crystallizing these complexes. We describe here procedures and approaches to prepare chromatin complexes, to crystallize chromatin complexes, and to improve diffraction properties through postcrystallization soaks. Special attention is paid to evaluating the quality of the purified chromatin complexes as well as assessing the presence of the chromatin protein or enzyme in crystals. The methods described for preparing and purifying chromatin complexes should be applicable to biochemical, biophysical, and other structural approaches including cryoelectron microscopy.
authors.affiliation: Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA, United States.
authors.forename: R K
authors.initials: RK
authors.lastname: McGinty
authors.affiliation: Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA, United States.
authors.forename: R D
authors.initials: RD
authors.lastname: Makde
authors.affiliation: Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA, United States. Electronic address: sxt30@psu.edu.
authors.forename: S
authors.initials: S
authors.lastname: Tan
mesh_terms: D000818:Animals; D002843:Chromatin / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods; D003460:Crystallization / Q000379:methods*; D004247:DNA / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry*
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D009707:Nucleosomes; D004247:DNA
keywords: Chromatin enzymes; Crystal dehydration; Fluorescent labeling; Macromolecular crystallization; Nucleosome core particle; Postcrystallization soaks; Protein–DNA complex; Size-exclusion chromatography