Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200kV

Abstract

SUMMARY A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.

Decision letter: 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy

“…We emphasize that we were able to obtain a T20S reconstruction at 2.98Å resolution using the full exposure of 53 e − /Å 2 and prior to weighting individual movie frames. We obtained similar results in determining the structure of an icosahedral virus to 3.7Å resolution using an exposure of 38 e − /Å 2 with a microscope operated at 200 kV (Campbell et al, 2014). These results contrast with previous measurements and demonstrate that useful high-resolution information may survive much higher exposures than previously thought for single particle cryoEM.…”

Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV

“…Several studies have used T. acidophilum 20S proteasome as a metric for characterizing the potential of TEMs and imaging accessories (2,14,16,29,39). We similarly sought to assess the resolution limit of the Talos Arctica using the 20S proteasome and two data collection strategies.…”

“…However, the high cost of purchasing and operating high-end TEMs renders such a setup unfeasible for many institutions. Campbell et al have previously reported the achievable resolution limit of an FEI TF20 TEM operating at 200 keV coupled with a DED to be up to ~3.7 Å (16). Recently, Li et al have determined the structure of a cypovirus capsid to ~3.3 Å using a Talos Arctica TEM (200 keV) with a DED, however, this molecule benefits from high molecular weight and very high internal symmetry (17).…”

Cryo-EM of bacterial pili and archaeal flagellar filaments

“…The great improvement in throughput provided by automated microscopes such as the Titan Krios allows for massive amounts of data to be collected in an unattended manner, but it has also been shown that one can reach a near-atomic resolution with direct electron detectors installed on more modest microscopes [10,11], albeit with much more effort. Similarly, advances in software for image analysis and reconstruction [12,13] have had an important effect, but people are still using legacy software such as SPIDER [14] to routinely reach near-atomic resolution, as evident from some of the papers that will be discussed.…”