Structural Superimposition

Abstact

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 micros 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.