title: Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM.
abstract: With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg2+and Zn2+. In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high-resolution reconstruction by single-particle cryo-EM.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, 10032.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York, 10032.
authors.forename: Cristina
authors.initials: C
authors.lastname: Gutierrez-Vargas
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York, 10032.
authors.forename: Jia
authors.initials: J
authors.lastname: Wei
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, 10032.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Grassucci
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York, 10032.
authors.forename: Ming
authors.initials: M
authors.lastname: Sun
authors.affiliation: Division of Infectious Diseases, New York State Department of Health, Wadsworth Center, Albany, New York, 12201.
authors.forename: Noel
authors.initials: N
authors.lastname: Espina
authors.affiliation: Division of Infectious Diseases, New York State Department of Health, Wadsworth Center, Albany, New York, 12201.
authors.forename: Susan
authors.initials: S
authors.lastname: Madison-Antenucci
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York, 10032.
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, 10032.|Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York, 10032.|Department of Biological Sciences, Columbia University, New York, New York, 10027.
authors.forename: Joachim
authors.initials: J
authors.lastname: Frank
mesh_terms: D014355:Chagas Disease; D020285:Cryoelectron Microscopy / Q000379:methods*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016053:RNA, Protozoan / Q000378:metabolism / Q000648:ultrastructure*; D012335:RNA, Ribosomal / Q000378:metabolism / Q000648:ultrastructure*; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*; D014349:Trypanosoma cruzi / Q000378:metabolism / Q000648:ultrastructure*; D015032:Zinc / Q000378:metabolism
chemical_list: D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D008274:Magnesium; D015032:Zinc
keywords: 2.5 Å resolution; Trypanosoma cruzi; high resolution cryo-EM; ribosome structure; single particle analysis