Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation.

Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether.

mRNA vaccine-induced IgG mediates nasal SARS-CoV-2 clearance in mice.

Coronavirus disease 2019 (COVID-19) mRNA vaccines that have contributed to controlling the SARS-CoV-2 pandemic induce specific serum antibodies, which correlate with protection. However, the neutralizing capacity of antibodies for emerging SARS-CoV-2 variants is altered. Suboptimal antibody responses are observed in patients with humoral immunodeficiency diseases, ongoing B cell depletion therapy, and aging.

H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.

SARS-CoV-2 and Other Coronaviruses in Rats, Berlin, Germany, 2023.

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.

Outcome of H5N1 clade 2.3.4.4b virus infection in calves and lactating cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel.

Microsecond time-resolved cryo-electron microscopy.

Microsecond time-resolved cryo-electron microscopy has emerged as a novel approach for directly observing protein dynamics. By providing microsecond temporal and near-atomic spatial resolution, it has the potential to elucidate a wide range of dynamics that were previously inaccessible and therefore, to significantly advance our understanding of protein function. This review summarizes the properties of the laser melting and revitrification process that underlies the technique and describes different experimental implementations.

Flash melting amorphous ice.

Water can be vitrified if it is cooled at high rates, which makes it possible to outrun crystallization in so-called no man's land, a range of deeply supercooled temperatures where water crystallizes rapidly. Here, we study the reverse process in pure water samples by flash melting amorphous ice with microsecond laser pulses. Time-resolved electron diffraction reveals that the sample transiently crystallizes despite a heating rate of more than 5 × 106 K/s, even though under the same conditions, vitrification can be achieved with a similar cooling rate of 107 K/s.

Shaped Laser Pulses for Microsecond Time-Resolved Cryo-EM: Outrunning Crystallization during Flash Melting.

Water vitrifies if cooled at rates above 3 × 10 K/s. In contrast, when the resulting amorphous ice is flash heated, crystallization occurs even at a more than 10 times higher heating rate, as we have recently shown. This may present an issue for microsecond time-resolved cryo-electron microscopy experiments, in which vitreous ice samples are briefly melted with a laser pulse because transient crystallization could potentially alter the dynamics of the embedded proteins.

Fast viral dynamics revealed by microsecond time-resolved cryo-EM.

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.

SARS-CoV-2 variants of concern elicit divergent early immune responses in hACE2 transgenic mice.

Knowledge about early immunity to SARS-CoV-2 variants of concern mainly comes from the analysis of human blood. Such data provide limited information about host responses at the site of infection and largely miss the initial events. To gain insights into compartmentalization and the early dynamics of host responses to different SARS-CoV-2 variants, we utilized human angiotensin converting enzyme 2 (hACE2) transgenic mice and tracked immune changes during the first days after infection by RNAseq, multiplex assays, and flow cytometry.

Omicron subvariant BA.5 efficiently infects lung cells.

The SARS-CoV-2 Omicron subvariants BA.1 and BA.2 exhibit reduced lung cell infection relative to previously circulating SARS-CoV-2 variants, which may account for their reduced pathogenicity.

Near-atomic resolution reconstructions from in situ revitrified cryo samples.

A microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in the liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged.

Electron diffraction of deeply supercooled water in no man's land.

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.

Assessment of Immunogenicity and Efficacy of CV0501 mRNA-Based Omicron COVID-19 Vaccination in Small Animal Models.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.

Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach.

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution. It involves melting a cryo sample with a laser beam to allow dynamics of the embedded particles to occur. Once the laser beam is switched off, the sample revitrifies within just a few microseconds, trapping the particles in their transient configurations, which can subsequently be imaged to obtain a snap shot of the dynamics at this point in time.

Visualizing Nanoscale Dynamics with Time-resolved Electron Microscopy.

The large number of interactions in nanoscale systems leads to the emergence of complex behavior. Understanding such complexity requires atomic-resolution observations with a time resolution that is high enough to match the characteristic timescale of the system. Our laboratory's method of choice is time-resolved electron microscopy.

Microsecond melting and revitrification of cryo samples: protein structure and beam-induced motion.

A novel approach to time-resolved cryo-electron microscopy (cryo-EM) has recently been introduced that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in the liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time resolution of just a few microseconds, this approach is notably fast enough to study the domain motions that are typically associated with the activity of proteins but which have previously remained inaccessible. Here, crucial details are added to the characterization of the method.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs. Here we show that the spike protein (S) from Alpha (also known as B.