# Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing

## TL;DR

This paper shows that LEA proteins can reduce sample damage during cryo-EM preparation, improving results and simplifying the process.

## Contribution

The novel use of LEA proteins as additives during plunge freezing to mitigate air-water interface damage in cryo-EM.

## Key findings

- LEA proteins reduce air-water interface damage during cryo-EM sample preparation.
- Cryo-electron tomography shows LEA proteins localize at interfaces, forming a protective barrier.
- High-resolution cryo-EM maps using LEA proteins are comparable to or better than existing methods.

## Abstract

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. In summary, we show LEA proteins can offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI- related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.