Laser Rehydration Restores Protein Structure After Vacuum Deposition for CryoEM

Cryogenic electron microscopy (cryoEM) has transformed structural biology, but its conventional sample preparation — blotting and plunge freezing — has persistent limitations. Proteins spend seconds at the air-water interface before vitrification, during which an estimated 90% adsorb to that interface, often denaturing or adopting preferred orientations that compromise 3D reconstruction quality. Native mass spectrometry (MS) soft-landing onto cryoEM grids has emerged as a promising alternative, bypassing the air-water interface entirely while also enabling gas-phase purification of complex mixtures. However, a critical obstacle remained: proteins deposited in vacuum lose water, causing measurable structural compaction that degrades resolution and distorts native conformation.

The Wisconsin team built upon their previously described cryo-landing instrument by integrating two new components: a molecular water doser that deposits amorphous ice at a calibrated rate of 1.2 nm/s, and a 532 nm continuous-wave laser modulated into 15 μs pulses via an acousto-optic modulator. After β-galactosidase ions were cryo-landed onto carbon-coated UltrAuFoil grids at ~12 pA ion current for 25 minutes, amorphous ice was deposited over the particles. The focused laser beam (~20 μm FWHM, 15–26 mW at the grid) was then systematically rastered across the grid surface, briefly liquefying the ice and allowing water molecules to rehydrate the compacted proteins before the thermal mass of the grid rapidly revitrified the sample. The entire post-laser transfer to liquid nitrogen was computer-controlled and completed in under one second.

CryoEM imaging on a 200 kV Glacios microscope revealed striking differences between conditions. Using map-to-model Fourier shell correlation (FSC) against PDB structure 6CVM at a 0.5 threshold, laser-rehydrated cryo-landed particles achieved 8.2 Å resolution compared to only 20 Å for non-lasered cryo-landed particles from the same grid — a 2.4-fold improvement. Conventional plunge-frozen particles reached 5.9 Å under more favorable imaging conditions (120,000× vs 73,000× magnification, smaller pixel size). Critically, to ensure fair comparison, all three reconstructions were computed using the same particle count of 5,684. The laser-rehydrated particles showed no detectable compaction and matched the known native quaternary structure of the β-galactosidase homotetramer.

The non-lasered cryo-landed particles, by contrast, exhibited the compaction previously documented by Rauschenbach et al. using molecular dynamics simulations — a collapse of the protein's hydration shell and tertiary contacts under vacuum. The laser rehydration step effectively reverses this damage by transiently restoring an aqueous environment around each particle before locking it back into vitreous ice. The 2D class averages and angular distribution maps for the rehydrated sample closely resembled those from plunge-frozen particles, indicating restored conformational homogeneity and more isotropic particle orientation.

The implications extend well beyond β-galactosidase as a model system. Because native MS can separate and select specific molecular species from heterogeneous mixtures — including cell lysates — this coupled MS-cryoEM pipeline could enable structural determination of proteins that cannot be purified by conventional biochemical means. The authors acknowledge that the current resolution (8.2 Å) still falls short of plunge freezing (5.9 Å), likely due to the lower magnification used for cryo-landed grids and the smaller particle dataset, rather than any fundamental limitation of the rehydration approach. Future optimization of ice thickness, laser parameters, and imaging conditions is expected to close this gap.