Published online 25 August 2016 Nucleic Acids Research, 2016, Vol. 44, No. 17 8013–8019 doi: 10.1093/nar/gkw708 NAR Breakthrough Article 3.9 ˚ A structure of the nucleosome core particle determined by phase-plate cryo-EM Eugene Y.D. Chua 1 , Vinod K. Vogirala 1 , Oviya Inian 1 , Andrew S.W. Wong 2 , Lars Nordenski ¨ old 1 , Juergen M. Plitzko 3 , Radostin Danev 3 and Sara Sandin 1,2,* 1 School of Biological Sciences, Nanyang Technological University, 637551, Singapore, 2 NTU Institute of Structural Biology, Nanyang Technological University, 639798, Singapore and 3 Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany Received April 22, 2016; Revised July 08, 2016; Accepted August 02, 2016 ABSTRACT The Volta phase plate is a recently developed elec- tron 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 deter- mine the structure of a small protein–DNA complex. To test the method, we made use of a 200 kDa Nu- cleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal struc- ture is known. We find that the phase plate provides a significant contrast enhancement that permits in- dividual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 ˚ A 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 im- portant method to determine novel near-atomic res- olution structures of small and challenging samples, such as nucleosomes in complex with nucleosome- binding factors. INTRODUCTION Single particle electron cryo-microscopy (cryo-EM) is a powerful technique for structure determination of isolated macromolecular complexes at near-atomic resolution. Sev- eral important developments have contributed to the recent ‘resolution revolution’ in cryo-EM, including direct electron detection, correction of beam-induced motion, as well as improved classifcation and 3D reconstruction procedures (1–3). The technique is particularly suitable for structure determination of large and fexible macromolecular com- plexes. Therefore, cryo-EM can potentially be used for high- resolution structural analysis of nucleosomes reconstituted in complex with chromatin-binding factors for which we have no or limited structural information. However, the nu- cleosome is a challenging target compared to proteins rou- tinely analysed in cryo-EM due to several reasons. First, nu- cleosomes are small (200 kDa), which can be problematic to detect (particle picking), align and structurally analyze (4). Second, excess DNA is required to prevent chromatin aggregation, resulting in a reduced contrast difference be- tween particles and the surrounding ice. Third, certain ori- entations of the nucleosomes are diffcult to detect, even in micrographs recorded at high defocus, resulting in system- atic loss of data and resolution. Therefore, interpretation of EM density and particle picking is problematic. Biological samples are weak-phase and radiation- sensitive objects (2,5). The most common approach to generate contrast in cryo-EM is to record images out of focus (defocus by 1–3 m), which is referred to as bright feld phase contrast EM. An alternative technique is to utilize phase plate EM (6). The principle is similar to phase contrast imaging in light microscopy with a Zernike phase plate. In a Transmission Electron Microscope (TEM), the phase plate is positioned in the back focal plane of the objective lens, introducing a phase shift between the scattered and un-scattered electron waves. The advantage of phase plate imaging over bright feld phase contrast imaging is that it maintains low-resolution frequencies without zeros or phase-fipping effects in the Contrast Transfer Function (CTF). Therefore, small particles can be detected and analysed without CTF correction. This was demonstrated in a recent phase-plate cryo-EM recon- * To whom correspondence should be addressed. Tel: +65 65923672; Fax: +65 67913856; Email: ssandin@ntu.edu.sg C  The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/nar/article/44/17/8013/2468058 by guest on 10 December 2022