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. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.
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. It is shown that single-particle reconstructions of apoferritin and Cowpea chlorotic mottle virus from revitrified samples are indistinguishable from those from conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by the instrument. How rapid revitrification affects the properties of the ice is also characterized, showing that revitrified samples exhibit comparable amounts of beam-induced motion. The results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and to address beam-induced specimen movement.
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. While we have previously performed such experiments with a modified transmission electron microscope, we here demonstrate a simpler implementation that uses an optical microscope. We believe that this will make our technique more easily accessible and hope that it will encourage other groups to apply microsecond time-resolved cryo-EM to study the fast dynamics of a variety of proteins.
We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in 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 domain motions that are typically associated with the activity of proteins, but which have previously remained inaccessible. Here, we add crucial details to the characterization of our method. We show that single-particle reconstructions of apoferritin and cowpea chlorotic mottle virus (CCMV) from revitrified samples are indistinguishable from those in conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by our instrument. We also characterize how rapid revitrification affects the properties of the ice, showing that revitrified samples exhibit comparable amounts of beam-induced motion. Our results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and address beam-induced specimen movement.SynopsisMicrosecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional 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. Two alternative implementations of the technique have previously been described, using either an optical microscope or performing revitrification experiments in situ. Here, it is shown that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within the spatial resolution. Interestingly, it is observed that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.
Proteins typically undergo conformational dynamics on the microsecond to millisecond timescale as they perform their function, which is much faster than the time-resolution of cryo-electron microscopy and has thus prevented real-time observations. Here, we propose a novel approach for microsecond time-resolved cryo-electron microscopy that involves rapidly melting a cryo specimen in situ with a laser beam. The sample remains liquid for the duration of the laser pulse, offering a tunable time window in which the dynamics of embedded particles can be induced in their native liquid environment. After the laser pulse, the sample vitrifies in just a few microseconds, trapping particles in their transient configurations, so that they can subsequently be characterized with conventional cryo-electron microscopy. We demonstrate that our melting and revitrification approach is viable and affords microsecond time resolution. As a proof of principle, we study the disassembly of particles after they incur structural damage and trap them in partially unraveled configurations.
We have recently introduced a microsecond time-resolved version of cryo-electron microscopy (cryo-EM) to enable the observation of the fast conformational motions of proteins. Our technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in 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. We have previously described two alternative implementations of the technique, using either an optical microscope or performing revitrification experiments in situ. Here, we show that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within our spatial resolution. Interestingly, we observe that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.
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