9y47

Publication Abstract from PubMed

Grid preparation is a rate-limiting step in determining high-resolution structures by single particle 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 straightforward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found the lipid affinity grids concentrate particles away from the AWI in thin ice ( approximately 30 nm). We determined three structures ranging from 2.4 to 3.0 A resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 A structure of a sub-100 kDa protein without symmetry, demonstrating the utility for a range of biological macromolecules. 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.

Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM.,Skrajna A, Lenger C, Robinson E, Cannon K, Sarsam R, Ouellette RG, Abotsi AM, Brennwald P, McGinty RK, Strauss JD, Baker RW J Struct Biol. 2025 Oct 11:108253. doi: 10.1016/j.jsb.2025.108253. PMID:41083086^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.