Abstract
During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Å from a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Å level to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.
Highlights
The studies of virus structures have played important roles in the historical development of cryo-electron microscopy
Ltd., Akishima, Tokyo, Japan) with non-negligible spherical aberration (Cs 3.3 mm) suited for developing phase-contrast imaging [42]. We refer to this setting as setting A, from which we obtained a cryo-electron microscopy (cryo-EM) structure of Dragon GNNV (DGNNV) at sub-4 Å
This structure was reported in regional conferences in the period of 2014–2015 but was soon shadowed by the X-ray structure (3.6 Å) [33] and the cryo-EM structure (3.9 Å) [43], obtained by a 300 kV highend cryo-EM with a CCD that precluded image de-blurring by motion correction [34]
Summary
The studies of virus structures have played important roles in the historical development of cryo-electron microscopy (cryo-EM). DeRosier and Crowther, at the Medical Research Council had established the mathematical principles for the reconstruction of three-dimensional structures from two-dimensional electron images using viruses with icosahedral symmetry [1,2]. A much more difficult route for non-periodical objects was pioneered by Hoppe [3] and eventually fulfilled by Frank in the establishment of the single-particle reconstruction as a general method to include proteins with low or no symmetry [4], paving the way for the cryo-EM resolution breakthrough. Several groups had endeavored to gradually extend the resolutions of cryo-EM for structure determination using icosahedral viruses [7,8,9], until the resolving of the main chain densities in the capsid protein using a high-end 300 kV cryo-EM with the images recorded on films [10]. Due to the ease of sample loading and screening, together with the high stability in electron beam quality and an excellent vacuum to support multiple days of data collection nonstop, this model soon proliferated in the wealthy regions of the world
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