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

Understanding protein function necessitates observing proteins in action. This study addresses the longstanding challenge of observing fast protein dynamics by employing a recently developed microsecond time-resolved cryo-EM technique. Cowpea chlorotic mottle virus (CCMV), a plant virus with an icosahedral capsid, serves as a model system. CCMV's capsid undergoes a significant conformational change—a pH-dependent swelling and contraction—crucial to its life cycle. Upon entering a host cell, CCMV experiences a change in pH and divalent ion concentration, triggering capsid expansion and subsequent RNA release. Conversely, lowering the pH below 5 causes the capsid to contract. Previous studies suggested complex large-scale motions within the capsid during this transition, but the speed and coordination of these movements remained unclear. Traditional time-resolved cryo-EM lacks the necessary temporal resolution to capture these fast dynamics, while ultrafast X-ray crystallography is not ideally suited to this system due to the constraints of crystallization. This research aims to overcome these limitations by applying the novel microsecond time-resolved cryo-EM technique to elucidate the detailed mechanics of CCMV capsid contraction.

The literature extensively details the structure and function of CCMV. The virus's ability to transition between contracted and expanded states, triggered by environmental pH changes, is well-documented. Studies suggest that this transition involves significant large-scale movements of capsid proteins, but the exact nature of these motions—whether concerted or asynchronous, and their timescale—remained elusive due to technological limitations. Existing techniques either lack the required time resolution or are unsuitable due to the limitations of crystallization. This gap in knowledge highlights the need for a method capable of capturing these fast conformational changes.

The researchers utilized a microsecond time-resolved cryo-EM approach. Cryo-samples of extended CCMV (at pH 7.6, without divalent ions) were prepared in the presence of a photoacid (NPE-caged-proton). UV irradiation (266 nm) released the photoacid, lowering the pH to 4.5. The vitreous ice matrix prevents immediate contraction. A laser beam (532 nm) rapidly melts the sample, initiating contraction. After 30 seconds, the laser is switched off, and the sample reverts to a vitrified state within microseconds, trapping the partially contracted particles for imaging. Single-particle reconstructions were obtained for the extended (pH 7.6), partially contracted (after melting and re-vitrification), and fully contracted (prepared at pH 5.0) states. The resolution of the reconstructions varied depending on the conformational heterogeneity of the samples, with the extended state showing the highest resolution (3.9 Å) and the partially contracted state having lower resolution (8.0 Å). Variability analysis using cryoSPARC software helped characterize the conformational heterogeneity of the partially contracted state, revealing a curved reaction path suggesting that different motions occurred on different timescales. Atomic models were docked into the reconstructions to analyze the translations and rotations of the capsid proteins in different stages of contraction. The analysis focused on the movements of the three subunits (A, B, and C) within the asymmetric unit of the icosahedral capsid.

The microsecond time-resolved cryo-EM revealed that the pH jump-induced contraction of the CCMV capsid occurs on a microsecond timescale. The contraction is a concerted process, but the movements of individual capsid proteins happen on slightly different timescales, resulting in a curved reaction pathway. The analysis of the protein subunit movements showed that the pentamers rotate about twice as fast as the hexamers, and the rotations of individual subunits within the asymmetric unit also occur on different timescales. The study observed a wide distribution in contraction speeds, likely due to factors such as variations in the temperature of the rehydrated area and the presence of three different virion types each packing a different RNA strand. The partially contracted state, captured using this technique, exhibits substantial conformational heterogeneity, making it difficult to attain very high resolutions. However, the variability analysis proved instrumental in distinguishing various stages of the contraction, unveiling the complex interplay of motions. The experiments confirmed the trapping effect of the vitreous ice matrix before laser melting, ensuring that the observed dynamics are triggered solely by the pH change and not by other factors.

This study successfully demonstrates the application of microsecond time-resolved cryo-EM to study fast, out-of-equilibrium protein dynamics. The detailed view of the CCMV capsid contraction obtained is unprecedented. The ability to capture transient, partially contracted configurations provides insight into the mechanism of this conformational change. The observed multi-timescale motions involved in the contraction, along with the significant conformational heterogeneity of the intermediate states, highlight the complexity of this process. The technique's success in visualizing these fast dynamics signifies a significant advancement in studying dynamic protein systems and in understanding biological processes far from equilibrium.

This work establishes the efficacy of microsecond time-resolved cryo-EM in studying fast protein dynamics. The detailed observation of CCMV capsid contraction reveals the multi-timescale nature of this process, highlighting the need for techniques that can resolve out-of-equilibrium dynamics. The method's general applicability, as suggested by the use of photoreactive compounds, opens avenues for investigating a wide array of protein systems and dynamic processes. Future studies could explore different stimuli, optimize experimental parameters, and apply this technique to other biological systems, potentially revealing even more intricate details about dynamic processes crucial for life.

The main limitation of the study lies in the resolution of the partially contracted state. The significant conformational heterogeneity of the partially contracted particles led to a lower resolution in the corresponding reconstruction compared to the extended and fully contracted states. This heterogeneity, although a natural aspect of the dynamic process, limits the level of atomic detail obtainable. Further refinement of the technique or use of other approaches might help resolve this issue and offer higher-resolution views of the intermediate states. The limited number of virion types used might not fully capture the variability of contraction speeds observed in other studies, although this is considered a minor factor due to the experimental approach that involves various samples and replicates. This method is still relatively new, thus, further optimizations may improve the resolution and broader application of this technique.