The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. Here, we combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed pre-fusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines.

Summary Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

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Cryo-electron tomography (cryo-ET) allows cellular ultrastructures and macromolecular complexes to be imaged in three-dimensions in their native environments. Cryo-electron tomograms are reconstructed from projection images taken at defined tilt-angles. In order to recover high-resolution information from cryo-electron tomograms, it is necessary to measure and correct for the contrast transfer function (CTF) of the microscope. Most commonly, this is performed using protocols that approximate the sample as a two-dimensional (2D) plane. This approximation accounts for differences in defocus and therefore CTF across the tilted sample. It does not account for differences in defocus of objects at different heights within the sample; instead, a 3D approach is required. Currently available approaches for 3D-CTF correction are computationally expensive and have not been widely implemented. Here we simulate the benefits of 3D-CTF correction for high-resolution subtomogram averaging, and present a user-friendly, computationally-efficient 3D-CTF correction tool, NovaCTF, that is compatible with standard tomogram reconstruction workflows in IMOD. We validate the approach on synthetic data and test it using subtomogram averaging of real data. Consistent with our simulations, we find that 3D-CTF correction allows high-resolution structures to be obtained with much smaller subtomogram averaging datasets than are required using 2D-CTF. We also show that using equivalent dataset sizes, 3D-CTF correction can be used to obtain higher-resolution structures. We present a 3.4 Å resolution structure determined by subtomogram averaging.