Detailed Preparation of Ni-NTA Lipid Monolayer Affinity Grids for Cryo-EM Structural Analysis of Soluble Polyhistidine-Tagged Protein Complexes.

Cryo‑specimen preparation remains a major technical challenge for high‑resolution structural determination of protein complexes. Interactions at the air‑water interface can induce protein denaturation or preferred orientation, thereby limiting achievable resolution. In addition, dilute samples or low‑abundance targets often exhibit insufficient particle density, which hinders efficient data collection. Lipid‑based affinity grids provide an effective strategy to address these limitations. This article presents a step‑by‑step protocol for preparing Ni‑NTA lipid monolayer affinity grids for the structural analysis of soluble polyhistidine‑tagged protein complexes. The major stages of the protocol include preparing a lipid mixture, casting the lipid mixture onto the surface of a reservoir buffer, transferring the resulting lipid monolayer onto TEM grids, optionally drying and storing the lipid grids for up to 1 week, and cryo‑plunging the lipid grids with the sample. Successful implementation of this protocol yields cryo‑grids containing a single layer of well‑dispersed particles with improved particle density relative to grids prepared without a lipid monolayer. The procedure can be completed within a few hours in a laboratory equipped with a cryo‑plunger, a chemical fume hood, and standard tools commonly used for cryo‑specimen preparation. By combining detailed written instructions with video demonstrations, this work aims to improve the accessibility, reproducibility, and broader adoption of Ni‑NTA lipid monolayer affinity grids within the cryo‑EM community.