Strategies for crystallizing a chromatin protein in complex with the nucleosome core particle.
The molecular details of how chromatin factors and enzymes interact with the nucleosome are critical to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resolution. These steps include both pre- and postcrystallization strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes containing nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystallization soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resolution.
Center for Eukaryotic Gene Regulation, Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Ravindra D
RD
Makde
Song
S
Tan
D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D003460:Crystallization / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity
D002843:Chromatin; D005656:Fungal Proteins; D006657:Histones; D019476:Insect Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes
Chromatin; Crystallization strategies; Macromolecular complex crystallization; Nucleosome; Protein–DNA interactions