Replacing H ₂ O with D ₂ O in cryo-EM reduces radiation damage in biological samples

Radiation damage limits the resolution achievable in electron microscopy of biological specimens. Hydrogen is the most abundant atom in these specimens, and its low mass maximizes the recoil energy it receives from incident electrons, making it the atom most readily lost under the beam. Substitution of hydrogen by deuterium halves this recoil energy and makes the bonds involved slightly harder to break, yet its protective effect on vitrified protein crystals has not been tested. Using cryogenic electron diffraction at 200 kV, we show that hen egg-white lysozyme crystals grown directly in D ₂ O withstand a dose 1.7 times higher than crystals grown in H ₂ O before their Bragg intensity at 10–7 Å falls to 1/ e (13.2 ± 1.0 against 7.7 ± 0.4 e ⁻ /Å ² ), with every D ₂ O-grown crystal outlasting every H ₂ O-grown crystal. The protection is achieved with deuteration confined mainly to the solvent and the exchangeable hydrogens at the protein surface, which form the hydrogen bonds that hold the crystal lattice together. The protein itself need not be deuterated by expression in deuterated media. Direct crystallization in D ₂ O involves only a single substitution in the mother liquor and offers an accessible strategy to extend the usable dose in electron diffraction of protein crystals.