Marcel Drabbels

Preferred particle orientation, which results from the adsorption of proteins to the air-water interface, is a frequently encountered issue in single-particle cryo-electron microscopy (cryo-EM). Particles then present only a limited number of views, which are oversampled during data acquisition at the expense of throughput, while other views may be missing altogether. In severe cases, a high-resolution reconstruction becomes unattainable. We have recently established that ultrasonic excitation of the sample during vitrification improves the angular distribution by detaching particles from the air-water interface and reshuffling their orientations. Here, we describe an implementation of this principle for a plunge freezing apparatus, in which the ultrasonic transducer is integrated into the head of the pair of tweezers holding the sample, and show that this design is able to reduce preferred orientation. We demonstrate this for hemagglutinin, the canonical example of preferred orientation, and show that the fraction of side and tilted views increases with the excitation amplitude. This allows us to obtain a complete reconstruction of the trimer at 2.5 Å.

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 deposit a layer of amorphous ice onto the sample and briefly flash melt it to rehydrate the soft-landed proteins. Melting with a 30 µs laser pulse creates a broad ensemble of partially rehydrated structures. However, a subset of particles returns to a configuration close to the native state. This allows us to propose strategies to fully rehydrate the entire ensemble and brings routine sample preparation by soft-landing mass spectrometry within reach.

Laser flash melting and revitrification experiments have recently improved the time resolution of cryo-electron microscopy (cryo-EM) to the microsecond timescale, making it fast enough to observe many of the protein motions that are associated with function. The technique has also opened up a new dimension for cryo-EM sample preparation, making it possible to deposit compounds onto a cryo-EM sample while it is frozen, so that upon flash melting, the embedded particles experience an altered environment. For example, we have recently shown that depositing ultrathin silicon dioxide membranes onto a cryo-EM sample causes particles to detach from the interface upon flash melting, removing preferred particle orientation. Here, we describe an apparatus for physical vapor deposition of compounds onto cryo-EM samples, detailing its design and operation. As a demonstration, we determine that the minimum thickness of silicon dioxide sealing membranes in a laser flash melting experiment is just over two monolayers. Moreover, we show that it is possible to deposit reagents onto the cryo-EM and induce mixing during flash melting, which points toward a new strategy for initiating protein dynamics in time resolved experiments. We propose that the design of our instrument can form the basis for an integrated platform for microsecond time-resolved cryo-EM experiments.

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.

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 µs. This allows us to observe the photoinduced dynamics of the light-driven sodium pump Er NaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.

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.

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-EM 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.

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 µs 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.

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.

The vitrification of aqueous solutions through rapid cooling is a remarkable achievement that launched the field of cryo-electron microscopy (cryo-EM) and has enabled the cryopreservation of biological specimens. For judging the feasibility of a vitrification experiment, the critical cooling rate of pure water is a frequently cited reference quantity. However, an accurate determination has remained elusive, with estimates varying by several orders of magnitude. Here, we employ time-resolved electron microscopy to obtain a precise measurement of this quantity. We use shaped microsecond laser pulses to briefly melt an amorphous ice sample before flash freezing it with a variable, well-defined cooling rate. This allows us to directly measure the critical cooling rate of water, which we determine to be 6.4 × 10 6 K / s . This result provides important insights into the question of how closely the conformational ensembles in plunge-frozen cryo samples reflect the structure of a protein at room temperature. Moreover, our experimental approach expands the toolkit of microsecond time-resolved cryo-EM, an emerging technique, in which a cryo sample is flash melted and revitrified with a laser pulse in order to enable time-resolved observations of protein dynamics.