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

Cowpea chlorotic mottle virus (CCMV) is an icosahedrally symmetric plant virus that must both securely package its RNA and release it at the right time to infect the host. Upon entering the host cytoplasm, the virus experiences a decrease in divalent ion concentration and an increase in pH, leading to capsid swelling (about 10% increase in diameter). Calcium ions normally complex with negatively charged residues on the capsid interior; their removal triggers electrostatic repulsion and expansion. In the absence of divalent ions, lowering the pH below 5 contracts the virus by protonating these residues and removing their repulsion. Despite structural snapshots of contracted and expanded states, the fast, large-scale translations and rotations underlying CCMV capsid mechanics have been unclear—specifically whether they occur concertedly or asynchronously and how fast they proceed. Prior work on a related icosahedral virus (Nudaurelia capensis ω) suggested pH-induced contraction completes by ~10 ms and may be faster, implying CCMV motions could be too fast for traditional millisecond time-resolved cryo-EM. There is thus a need for an approach with microsecond time resolution to directly observe these rapid, non-equilibrium dynamics.

The authors reference comparisons between contracted and expanded virus structures indicating multiple large-scale protein motions are involved in capsid mechanics, but their timing and coordination remained unresolved. For another icosahedral virus (Nudaurelia capensis ω), pH-induced contraction was complete after ~10 ms but likely faster, highlighting limitations of traditional millisecond time-resolved cryo-EM to capture sub-millisecond events. Ultrafast X-ray crystallography can achieve sufficient temporal resolution but is unsuitable for these dynamics in crystals. Methods such as conventional cryo-EM at equilibrium cannot access transient, partially contracted intermediates. Advances in computational analyses (e.g., cryoSPARC) enable variability analysis but require time-resolved data to connect structural heterogeneity with reaction pathways. This body of literature motivated the development and application of microsecond time-resolved cryo-EM to capture fast, out-of-equilibrium protein motions.

- Sample preparation: CCMV particles were prepared in the extended state at pH 7.6 without divalent ions and embedded in vitreous ice in the presence of a photoacid (NPE-caged-proton). - Chemical trigger (pH jump): The pH of the cryo sample was lowered to 4.5 by UV irradiation at 266 nm to uncage protons from the photoacid. In vitreous ice, the particles could not respond and remained in the extended configuration. - Time-resolved initiation: A green laser (532 nm) rapidly melted the cryo sample. Once liquid, capsids began contracting in response to the previously induced pH jump. - Rapid quench: The heating laser was switched off, causing the thin film to cool and revitrify within microseconds, trapping particles in partially contracted states corresponding to defined time windows after melting. - Controls and reference states: Reconstructions were obtained for (i) extended CCMV (pH 7.6), (ii) fully contracted CCMV prepared separately at pH 5.0, and (iii) partially contracted CCMV trapped by melt–revitrification after the pH jump. A reconstruction of extended CCMV following pH reduction in vitreous ice (without melting) was indistinguishable from the extended control, confirming that ice immobilization prevents contraction. - Cryo-EM data and analysis: Single-particle reconstructions were performed, including extended (3.9 Å resolution), partially contracted (reported at ~8.0 Å; heterogeneity-limited), and contracted states. Variability analysis (cryoSPARC) was conducted on particle sets from extended, intermediate, and contracted ensembles. The first variability component corresponded predominantly to particle diameter changes; the second, to capsid protein motions. Particles were binned into 30 slices along the first component, and reconstructions were computed for each slice. Atomic models of extended and contracted states were docked into slices to extract per-subunit translations and rotations, and particle diameters were measured along the five-fold symmetry axis. Rotational analyses quantified pentamer and hexamer rotations and subunit-specific angles for A, B, and C within the asymmetric unit.

- Time scale: Capsid contraction of CCMV proceeds on the microsecond timescale once the sample is melted. Some particles complete a substantial fraction (about half) of the contraction within the laser-pulse time window reported in the experiments. - Concerted yet asynchronous motions: Contraction is overall concerted but composed of protein translations and rotations that occur on different timescales, producing a curved reaction path in variability space. - Particle size changes: Extended capsid diameter ~32 nm; fully contracted diameter ~28 nm; partially contracted states centered near ~31 nm, spanning a broad continuum between extended and contracted sizes. - Heterogeneity: The melt–revitrified ensemble exhibits substantial conformational heterogeneity, limiting attainable resolution for the intermediate reconstruction and showing a wide distribution of diameters across slices 6–12 (with slice 12 approximately halfway between extended and contracted diameters). - Rotational mechanics: Contraction is accompanied by simultaneous clockwise rotations of pentamers and hexamers, each by about 5° in the fully contracted state. Pentamers rotate approximately twice as fast as hexamers across the reaction coordinate. - Subunit-specific kinetics: Within the asymmetric unit (subunits A, B, C), subunit A reaches its final rotation angle (~−7°) earlier (by slice 12), whereas B and C are only about halfway to their final rotations (~−11° and ~−13°), revealing distinct timescales for subunit motions. - Variability analysis: Extended, intermediate, and contracted conformations form distinct clusters in the first two variability components, with the reaction path curved and extended/partially contracted ensembles partially overlapping. - Mechanistic interpretation: Removal of electrostatic repulsion upon pH jump triggers a rapid, dissipative collapse, with a broad spread in contraction velocities likely due to dissipative dynamics, virion-to-virion differences (three RNA-containing species), and local temperature variations during rehydration.

The study answers the central question of whether microsecond time-resolved cryo-EM can capture fast, non-equilibrium protein motions by directly visualizing CCMV capsid contraction after a pH jump. The results show that capsid contraction occurs on microsecond timescales and involves coordinated but temporally distinct translations and rotations among capsid subunits, which could not be resolved with equilibrium methods or slower time-resolved approaches. Variability analysis maps a curved trajectory linking extended, intermediate, and contracted conformations, indicating multiple coupled motions with differing kinetics. These observations validate the method’s ability to trap transient intermediates via melt–revitrification, revealing mechanistic details such as pentamer/hexamer rotations and subunit-specific timing. The work suggests broad applicability of microsecond time-resolved cryo-EM to other rapid protein dynamics in out-of-equilibrium conditions, overcoming longstanding temporal limitations in structural biology.

Microsecond time-resolved cryo-EM, enabled by photochemical triggering in vitreous ice followed by rapid melting and revitrification, reveals the fast, concerted yet asynchronous mechanics of CCMV capsid contraction. The method captures transient, partially contracted intermediates, quantifies diameter changes and subunit-specific rotations, and delineates a curved reaction path across variability space. Beyond elucidating CCMV mechanics, the approach establishes a general framework for initiating and trapping rapid conformational changes using photoreleased stimuli. It should extend to diverse biological processes using caged ions, small molecules, ATP, amino acids, or peptides, offering a path to observe fast non-equilibrium protein dynamics previously inaccessible to structural methods. Future work can refine temporal control, reduce heterogeneity, and apply the technique to other complex assemblies.

- Conformational heterogeneity in the melt–revitrified intermediate ensemble limited reconstruction resolution and complicated precise kinetic assignment. - A broad spread in contraction velocities was observed, likely due to dissipative dynamics, differences among the three CCMV virion species (packing different RNA strands), and local temperature variations during the melt–revitrification process. - While the method traps intermediates at microsecond scales, the exact timing window depends on laser parameters and heat transfer, introducing variability across particles. - Conventional equilibrium reconstructions cannot capture these transient states, necessitating specialized instrumentation and protocols (photoacids, UV triggering, laser melting), which may not be universally available.