Whole-image contrast transfer function (CTF) estimation was performed using CTFFind4 11 and micrographs yielding CTF estimates below an Appion confidence value of 0.9 were discarded. Mechanical and beam-induced motions were corrected and dose weighting performed using MotionCor2 10. To minimize the effects of electron beam-induced motions, samples were prepared on gold grids 9 and exposures were acquired at the center of holes (1.2 Ојm diameter) using a beam (~2 Ојm diameter) large enough to cover the entirety of the hole and contact the surrounding gold substrate. Images were acquired at a nominal magnification of 45,000Г— (calibrated pixel size of 0.91 Г… at detector level) with the Gatan K2 Summit DED operating in super-resolution mode. Data were collected using Leginon 7 and image pre-processing was performed using the Appion pipeline 8. 6 immediately prior to data collection, as implemented in Leginon 7.

Coma-free alignment was performed according to Glaeser et al.

Careful alignment of the TEM was performed before each data collection to maximize parallel illumination 6 (see Methods and Protocol Exchange DOI) ( Supplementary Figure 1) and ensure Thon rings were visible beyond ~3 Г… resolution in the power spectrum of images collected over amorphous carbon. Our work represents a significant advancement in the attainable resolution limit for structures obtained using a 200 keV TEM, proving that such TEMs equipped with a DED are capable of reconstructing macromolecules of varying sizes and symmetries at resolutions previously ascribed only to 300 keV microscopes.ДЄll data were acquired using a base-model Talos Arctica TEM operating at 200 keV. The resulting maps are of sufficient quality to identify ordered water molecules and unambiguously assign amino acid rotameric conformations. Here we expand upon prior studies utilizing 200 keV TEMs 4, 5, and demonstrate that a Thermo Fisher Talos Arctica (200 keV) paired with a Gatan K2 Summit can produce cryo-EM reconstructions of Thermoplasma acidophilum 20S proteasome and rabbit muscle aldolase at ~3.1 Г… and ~2.6 Г… resolution, respectively. However, the high cost of purchasing and operating high-end TEMs may be unfeasible for many institutions. TEMs operating at 300 keV offer minimized inelastic scattering and specimen charging over those at lower voltages, the benefits of which confer an advantage in imaging thicker specimens 3. To date, the vast majority of high-resolution cryo-EM reconstructions have been obtained using transmission electron microscopes (TEMs) operating at 300 keV equipped with a direct electron detector (DED). Recent technical breakthroughs in the field of three-dimensional single-particle cryo-electron microscopy (cryo-EM) have allowed for the visualization of biological macromolecular assemblies in near-native states at unprecedented resolutions 1, 2.

L 3 the talos principle image