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We inoculated 6 cattle with severe acute respiratory syndrome coronavirus 2 and kept them together with 3 uninoculated cattle. We observed viral replication and specific seroreactivity in 2 inoculated animals, despite high levels of preexisting antibody titers against a bovine betacoronavirus. The in-contact animals did not become infected.

Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable such observations, we have recently proposed a technique that improves the time resolution of cryo-electron microscopy (cryo-EM) to microseconds. Here, we demonstrate that microsecond time-resolved cryo-EM enables observations of fast protein dynamics. We use our approach to elucidate the mechanics of the capsid of cowpea chlorotic mottle virus (CCMV), whose large-amplitude motions play a crucial role in the viral life cycle. We observe that a pH jump causes the extended configuration of the capsid to contract on the microsecond timescale. While this is a concerted process, the motions of the capsid proteins involve different timescales, leading to a curved reaction path. It is difficult to conceive how such a detailed picture of the dynamics could have been obtained with any other method, which highlights the potential of our technique. Crucially, our experiments pave the way for microsecond time-resolved cryo-EM to be applied to a broad range of protein dynamics that previously could not have been observed. This promises to fundamentally advance our understanding of protein function. Here, the authors show that microsecond time-resolved cryo-EM can be used to observe real-life protein dynamics, which they demonstrate by capturing the pH-induced contraction of the CCMV capsid.

We tested 130 rats captured in Berlin for coronaviruses. SARS-CoV-2 antibodies were detected in 1 rat, but all animals were negative by reverse transcription PCR, suggesting SARS-CoV-2 was not circulating in the rat population. However, alphacoronaviruses were found. Monitoring rodent populations helps to determine coronavirus occurrence, transmission, and zoonotic potential.

Protein structure determination and prediction have made stunning progress. In contrast, it is generally not possible to observe proteins as they perform their tasks, which leaves our understanding of these nanoscale machines fundamentally incomplete. My group has recently introduced a novel approach to time-resolved cryo-EM that improves its time resolution by about 3 orders of magnitude, making it fast enough to observe the microsecond dynamics of proteins that are frequently associated with function. Our method involves melting a cryo sample with a laser beam, which allows dynamics of the embedded particles to occur in liquid if a suitable stimulus is provided. When the heating laser is switched off, the sample rapidly revitrifies, trapping the particles in their transient configurations, in which we can subsequently image them. As I will illustrate, this makes it possible to watch protein dynamics that were previously unobservable. Our method therefore promises to fundamentally advance our understanding of protein function. We also demonstrate that laser melting and revitrification of cryo samples can be used to overcome preferred particle orientation, an issue that still causes many cryo-EM projects to fail. Finally, I will show a new approach that significantly expands the utility of our method by extending its temporal observation window. This allows us to follow the structural evolution of conformational ensembles in a temperature jump experiment on a timescale of hundreds of microseconds.

After experimental inoculation, severe acute respiratory syndrome coronavirus 2 infection was confirmed in bank voles by seroconversion within 8 days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Thus, bank voles are unlikely to establish effective transmission cycles in nature.

A generally accepted understanding of the anomalous properties of water will only emerge if it becomes possible to systematically characterize water in the deeply supercooled regime, from where the anomalies appear to emanate. This has largely remained elusive because water crystallizes rapidly between 160 K and 232 K. Here, we present an experimental approach to rapidly prepare deeply supercooled water at a well-defined temperature and probe it with electron diffraction before crystallization occurs. We show that as water is cooled from room temperature to cryogenic temperature, its structure evolves smoothly, approaching that of amorphous ice just below 200 K. Our experiments narrow down the range of possible explanations for the origin of the water anomalies and open up new avenues for studying supercooled water. Supercooled water in so-called “no man’s land” promises to reveal the origin of the water anomalies. Here, the authors use electron diffraction to provide the first characterization that spans this temperature range, which narrows down the array of possible explanations.

Microsecond time-resolved cryo-electron microscopy promises to significantly advance our understanding of protein function by rendering cryo-electron microscopy (cryo-EM) fast enough to observe proteins at work. This emerging technique involves flash melting a cryo sample with a laser beam to provide a brief time window during which dynamics are initiated. When the laser is switched off, the sample revitrifies, arresting the proteins in their transient configurations. However, observations have so far been limited to tens of microseconds only, due to the instability of the thin liquid film under laser irradiation. Here, we seal samples between two ultrathin, vapor-deposited silicon dioxide membranes to extend the observation window by an order of magnitude. These membranes not only allow for reconstructions with near-atomic spatial resolution, but can also be used to eliminate preferred particle orientation. We showcase our technology by performing a time-resolved temperature jump experiment on the 50S ribosomal subunit that provides new insights into the conformational landscape of the L1 stalk. Our experiments significantly expand the capabilities of microsecond time-resolved cryo-EM and promise to bridge the gap to the millisecond timescale, which can already be addressed with traditional approaches. Laser flash melting experiments have rendered cryo-EM fast enough to observe the microsecond motions of proteins. The authors extend the observation window of this emerging technique tenfold, to hundreds of microseconds, by sealing cryo-EM samples in ultrathin liquid cells.

Nanofluidics involves the study of fluid transport in nanometer-scale structures. We report the direct observation of fluid dynamics in a single zinc oxide nanotube with the high spatial and temporal resolution of four-dimensional (4D) electron microscopy. The nanotube is filled with metallic lead, which we melt in situ with a temperature jump induced by a heating laser pulse. We then use a short electron pulse to create an image of the ensuing dynamics of the hot liquid. Single-shot images elucidate the mechanism of irreversible processes, whereas stroboscopic diffraction patterns provide the heating and cooling rates of single nanotubes. The temporal changes of the images enable studies of the viscous friction involved in the flow of liquid within the nanotube, as well as studies of mechanical processes such as those that result in the formation of extrusions.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies. Emerging SARS-CoV-2 variants with mutations in the spike protein raise concerns regarding vaccine efficacy. Here, the authors show that two spike encoding mRNA vaccines in preclinical and clinical development protect human ACE2 mice from the B.1.351 variant of concern and ancestral B BavPat1.