title: Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.
abstract: Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method.
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Sandip
authors.initials: S
authors.lastname: Kaledhonkar
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Sun
authors.affiliation: Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
authors.forename: Bingxin
authors.initials: B
authors.lastname: Shen
authors.affiliation: Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Zonghuan
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Wadsworth Center, Albany, New York State Department of Health, Albany, NY 12201, USA.
authors.forename: David
authors.initials: D
authors.lastname: Barnard
authors.affiliation: Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Toh-Ming
authors.initials: TM
authors.lastname: Lu
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Gonzalez
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. Electronic address: jf2192@cumc.columbia.edu.
authors.forename: Joachim
authors.initials: J
authors.lastname: Frank
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D054657:Ribosome Subunits / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure*; D013997:Time Factors