Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM

Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryogenic electron microscopy (cryo-EM). Particle interaction with the air-water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air-water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the “resolution revolution” of cryo-EM. Here, we provide a straight-forward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found that lipid affinity grids concentrate particles away from the air-water interface in thin ice (∼30 nm). We determined a 2.6 Å structure of the human nucleosome, showing this method is amenable to high-resolution structure determination. Furthermore, we determined a 3.1 Å structure of a sub-100 kDa protein demonstrating that this technique is amenable to proteins across biological size ranges. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air-water interface, and thicker ice.