CryoEM at 100keV: A demonstration and prospects

Abstract

100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300Å in thickness, the predicted optimal electron energy for imaging is 100keV. Currently available electron cryomicroscopes in the 100-120keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100keV electrons. To demonstrate the potential of imaging at 100kV, the voltage of a standard, commercial 200kV field-emission gun (FEG) microscope was reduced to 100kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5MDa to 64kDa with corresponding diameters from 320 to 72Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.