Initial datasets were collected for sample quality control and low-resolution ribosome reconstruction on a 120 keV Tecnai Spirit cryo-EM (FEI; MPI Molecular Genetics, Berlin) equipped with a CMOS camera (TVIPS), with automated Leginon software (Carragher et al., 2000 (link); Suloway et al., 2005 (link)). Projection images were then analyzed by 3-D reconstruction and unsupervised classification for intrinsic ribosomal structure heterogeneity in silico with SPIDER (Frank et al., 1996 (link)) as described previously (Behrmann et al., 2015 (link); Loerke et al., 2010 (link)). These data revealed the presence of extra-ribosomal density at the 60S exit tunnel. To validate these findings, an independent biological replicate sample was re-prepared, with new grids frozen, and likewise imaged using the same protocol, yielding identical density at the 60S exit tunnel.
High-resolution data were collected on a 300 keV Titan Krios (FEI; EMBL, Heidelberg; Diamond Light Source, Oxfordshire) equipped with a Gatan Quantum K2 direct electron detector at 103.000x magnification, yielding a pixel size of 0.66 Å on the object scale. Movie stacks were collected in super-resolution mode with SerialEM (Mastronarde, 2005 (link)) with the following parameters: defocus range of 0.5-2.5 μm, 40 frames per movie, 20 s exposure time, electron dose of 1.589 e/Å2/s and a cumulative dose of 31.78 e/Å2 per movie.
High-resolution data were collected on a 300 keV Titan Krios (FEI; EMBL, Heidelberg; Diamond Light Source, Oxfordshire) equipped with a Gatan Quantum K2 direct electron detector at 103.000x magnification, yielding a pixel size of 0.66 Å on the object scale. Movie stacks were collected in super-resolution mode with SerialEM (Mastronarde, 2005 (link)) with the following parameters: defocus range of 0.5-2.5 μm, 40 frames per movie, 20 s exposure time, electron dose of 1.589 e/Å2/s and a cumulative dose of 31.78 e/Å2 per movie.