Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 Å resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABA _A receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 Å for apoferritin, 2.7 Å for the 20S proteasome, 2.8 Å for the GABAA receptor, 5.0 Å for haemoglobin, and 3.5 Å for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 Å. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.