title: 3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM.
abstract: The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.
authors: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors: Eugene Y D
authors: EY
authors: Chua
authors: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors: Vinod K
authors: VK
authors: Vogirala
authors: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors: Oviya
authors: O
authors: Inian
authors: NTU Institute of Structural Biology, Nanyang Technological University, 639798, Singapore.
authors: Andrew S W
authors: AS
authors: Wong
authors: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors: Lars
authors: L
authors: Nordenskiöld
authors: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors: Juergen M
authors: JM
authors: Plitzko
authors: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors: Radostin
authors: R
authors: Danev
authors: School of Biological Sciences, Nanyang Technological University, 637551, Singapore NTU Institute of Structural Biology, Nanyang Technological University, 639798, Singapore ssandin@ntu.edu.sg.
authors: Sara
authors: S
authors: Sandin
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D004247:DNA / Q000648:ultrastructure; D009707:Nucleosomes / Q000648:ultrastructure*; D014982:Xenopus laevis
chemical_list: D009707:Nucleosomes; D004247:DNA