Diversifying particle-orientation distributions in cryo-EM with LEA protein additives.

Preferential orientation at the air-water interface (AWI) remains a persistent challenge in cryo-electron microscopy, often requiring extensive optimization or specialized grid preparation strategies. Here, we show that late embryogenesis abundant (LEA) proteins, previously shown to protect fragile samples from AWI-induced damage, can be leveraged to resolve sample orientation bias. The addition of LEA proteins can yield either new orientation distributions or more isotropic ones in samples suffering from orientation biases. These findings establish LEA proteins as a practical class of cryo-EM additives that can reduce AWI-induced damage and improve particle-orientation distributions in single-particle analysis.