Biology subjects

Hruby, J.

Publications and source records attributed to Hruby, J..

5 recordsLinked to original sources

bioRxiv2025

Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification

Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the fast timescales on which they perform their tasks. Recently, laser flash melting and revitrification experiments have improved the time resolution of cryo-electron microscopy (cryo-EM) to microseconds, rendering it fast enough to observe the domain motions of proteins that are frequently linked to function. However, observations have been limited to a time window of just a few hundred microseconds. Here, we introduce time-resolved cryo-EM experiments based on jet vitrification that combine microsecond resolution with an observation window of up to seconds. We use a short laser pulse to initiate protein dynamics, and as they unfold, vitrify the sample with a jet of a liquid cryogen to arrest the dynamics at that point in time. We demonstrate that our approach affords near-atomic spatial resolution and a time resolution of 21 {micro}s. This allows us to observe the photoinduced dynamics of the light-driven sodium pump ErNaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.

biophysics↗

bioRxiv2025

Overcoming Preferred Orientation in Cryo-EM With Ultrasonic Excitation During Vitrification

Preferred particle orientation remains a frequently encountered problem in cryo-electron microscopy that arises when proteins adsorb to the air-water interface in only a limited number of orientations. This issue can significantly increase the data acquisition time required to reach a desired resolution or even make it impossible to obtain a reconstruction altogether. Here, we show that preferred orientation can be overcome by continuously exciting the sample with ultrasonic waves during vitrification. Our experiments suggest that mechanical oscillations induced in the sample support continuously shake proteins loose from the air-water interface, thereby scrambling their orientations. The simple, physical nature of this mechanism should make it applicable to a wide range of proteins. Since our method can be easily implemented in existing vitrification devices, we expect it to find widespread adoption.

biophysics↗

bioRxiv2025

Cryo-EM Sample Preparation with Soft-Landing and Laser Flash Melting

The preparation of cryo-EM samples by soft-landing mass spectrometry promises to significantly simplify sample optimization, which has remained an important bottleneck in single-particle cryo-electron microscopy (cryo-EM). However, only compacted configurations are observed after soft-landing since the proteins are dehydrated in the process. Here, we demonstrate that proteins can be returned to their native state by depositing a layer of amorphous ice and briefly flash melting the sample to rehydrate the soft-landed proteins. Melting with a 30 {micro}s laser pulse creates a broad ensemble of partially rehydrated conformations. However, a subset of particles recovers their native configuration. This allows us to propose strategies to fully rehydrate the entire ensemble and brings routine sample preparation by soft-landing mass spectrometry within reach. Finally, the experiments also expand the toolbox of microsecond time-resolved cryo-EM for initiating a broader range of protein dynamics.

biophysics↗

bioRxiv2025

Ultrathin Liquid Cells for Microsecond Time-Resolved Cryo-EM

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. Finally, we perform 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.

biochemistry↗

bioRxiv2024

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. We have previously observed that laser flash melting and revitrification of cryo samples reduces preferred orientation for large, symmetric particles. Here, we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.

biophysics↗