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.

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.

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.

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.

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.

Microsecond melting and revitrification of cryo samples.

The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase.