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 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.