Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.

Preparation of time-resolved cryo-EM grids Quantifoil R1.2/1.3 300 mesh Cu EM grids were carbon-coated and glow-discharged following standard procedures ( Grassucci et al., 2007 ). The ribosome recycling reaction was performed using a mixing-spraying device ( Lu et al., 2009 ) with an environmental chamber as previously described (Chen et al.) with a few minor alterations. During the experiment, the ambient conditions were maintained at 24 – 26 °C and 80% – 90% relative humidity. In the mixing-spraying chips equal volumes of two mixtures were injected, each at a flow rate of 3 μl per second. Materials Ribosomes ( E. coli MRE600) and mRNA, encoding the peptide fMet-Phe-Thr, were prepared as described previously( Borg et al., 2015 ). His-tagged EF-G and RRF were overexpressed in E. coli and purified by nickel affinity chromatography. tRNA Phe was overexpressed in and purified from E. coli . All experiments were performed in polymix buffer, containing 95 mM KCl, 5 mM NH 4 Cl, 0.5 mM CaCl 2 , 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate (pH 7.5), 1 mM dithioerythritol and 5 mM Mg(OAc) 2 . All reaction mixtures also contained components for energy supply GTP (1 mM), ATP (1 mM), phosphoenolpyruvate (PEP, 10 mM), pyruvate kinase (PK, 50 μg/ml), myokinase (MK, 2 μg/ml). Control experiment A post-termination complex mixture was prepared containing 70S ribosomes (2 μM), RRF (90 μM), tRNA Phe (5 μM) and MFT mRNA (10 μM). A recycling mixture was prepared containing nothing but polymix buffer and components for energy regeneration. The two mixtures were incubated for 15 min at 37 °C and then kept on ice. They were then centrifuged for 3 min at 20,800×g before being loaded into the mixing-spraying device. Equal volumes of the two mixtures were rapidly mixed in a reaction chip with 560 ms total incubation time from mixing to freezing on the EM grid. Time resolved cryo-EM experiment A post-termination complex mixture was prepared containing 70S ribosomes (2 μM), RRF (90 μM), tRNA Phe (5 μM) and MFT mRNA (10 μM). A recycling mixture was prepared containing IF3 (16 μM), and EF-G (20 μM). The two mixtures were incubated for 15 min at 37 °C and then kept on ice. They were centrifuged for 3 min at 20,800×g before being loaded into the mixing-spraying device. Equal volumes of the two mixtures were rapidly mixed in a reaction chip with 140 ms total incubation time from mixing to freezing on the EM grid. In long-incubation experiment, equal volumes of the two mixtures were mixed and incubated for 25 min at 25 °C. Then the reaction mixture was split into two halves and loaded into the mixing-spraying device. The EM grid was made in the same way as in 140 ms time-resolved cryo-EM experiment. The total reaction time was roughly estimated to be 30 min (25 min incubation and 5 min preparation of EM grid with mixing-spraying device).

Data Acquisition

The grids were imaged using a FEI Tecnai Polara transmission electron microscope operated at 300 kV and at a nominal magnification of ×31,000. Data sets were collected with Leginon( Potter et al., 1999 ) on a K2 Summit direct electron detector (Gatan, Pleasanton, CA) with a physical pixel size of 5 μm, corresponding to 1.255 Å per pixel at the specimen in electron counting mode. The dose rate was set to eight counts per physical pixel per second. The total exposure time was 10 s. 50 frames were recorded. All images were taken with a defocus in the range of 1.5 – 3 μm.

Image Processing

For the control experiment data set, after assessment of the micrographs, 963 micrographs were selected for subsequent processing. For the 140 ms experiment and long-incubation data set, 947 and 881 micrographs were selected for subsequent processing, respectively. To correct for stage movement and beam-induced movements, all 50 frames for each micrographs were aligned (Li et al., 2013). Particle picking was done with Relion 1.3( Scheres, 2012 ) and manually checked. For control experiment, all picked particles were classified into four classes. One class did not conform to the known shapes of ribosomes were rejected. Good classes were regrouped and reconstructed with auto-refinement in Relion. For 140 ms experiment, classification was done in four steps. In a first step, it is performed with low-pass-filtered reference maps and four-times binned particle images to separate the data into 30S subunits, 50S subunits and 70S ribosomes. The next level of classification resolved conformational differences, and two classes were obtained, “non-rotated 70S” (NR 70S) and “rotated 70S” (RT 70S), based on the presence or absence of intersubunit rotation. The RT 70S class was further divided into two subclasses. The RT 70S class was further divided into two subclasses, one (PostTC•RRF 140 ) identical (save for the difference in resolution) to the control complex (PostTC•RRF control ), and the other (PostTC•RRF•EF-G 140 ). Using focused classification, by applying a soft-edged mask to the EF-G and RRF-binding region, we found no evidence of subclasses in PostTC•RRF•EF-G 140 . The 50S subunit class was resolved into two subclasses of RRF- and EF-G-containing 50S subunits, distinct by the presence or absence of an E-site tRNA (“50S•RRF•EF-G•tRNA 140 ” and “50S•RRF•EF-G 140 ”). The 30S subunit class was resolved into two subclasses, one bound with tRNA in the P/I position (“30S•tRNA 140 ”) and the other with IF3 (“30S•IF3 140 ”). For long-incubation experiment, in the first step of classification, we found two classes, 50S and 30S subunits. In the next level, regrouped 30S subunits or 50S subunits were classified into subclasses. All the 30S subunit was found to be bound with IF3. For 50S subunits, four classes were found, 50S•RRF•EF-G long , 50S•RRF•EF-G•tRNA long , 50S•RRF•tRNA long and 50S•tRNA long .

Materials Ribosomes ( E. coli MRE600) and mRNA, encoding the peptide fMet-Phe-Thr, were prepared as described previously( Borg et al., 2015 ). His-tagged EF-G and RRF were overexpressed in E. coli and purified by nickel affinity chromatography. tRNA Phe was overexpressed in and purified from E. coli . All experiments were performed in polymix buffer, containing 95 mM KCl, 5 mM NH 4 Cl, 0.5 mM CaCl 2 , 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate (pH 7.5), 1 mM dithioerythritol and 5 mM Mg(OAc) 2 . All reaction mixtures also contained components for energy supply GTP (1 mM), ATP (1 mM), phosphoenolpyruvate (PEP, 10 mM), pyruvate kinase (PK, 50 μg/ml), myokinase (MK, 2 μg/ml).

Supplementary Material supplement