title: Structural snapshots of actively translating human ribosomes.
abstract: Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Behrmann
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Justus
authors.initials: J
authors.lastname: Loerke
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Budkevich
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Kaori
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Institut für Medizinische Physik und Biophysik, AG Protein X-Ray Crystallography, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical School, 6431 Fannin MSB 6.220, Houston, TX 77054, USA.
authors.forename: Pawel A
authors.initials: PA
authors.lastname: Penczek
authors.affiliation: FEI Company, Nanoport Europe, Achtseweg Noord 5, 5651 GG Eindhoven, the Netherlands.
authors.forename: Matthijn R
authors.initials: MR
authors.lastname: Vos
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bürger
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Max-Planck Institut für Molekulare Genetik, Ihnestraße 63-73, 14195 Berlin, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mielke
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Institut für Medizinische Physik und Biophysik, AG Protein X-Ray Crystallography, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Scheerer
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address: christian.spahn@charite.de.
authors.forename: Christian M T
authors.initials: CM
authors.lastname: Spahn
mesh_terms: D000595:Amino Acid Sequence; D020285:Cryoelectron Microscopy; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure*; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D012343:RNA, Transfer