9Y48

Sro7 bound to His-Exo84 (1-326) on a Nickel-NTA lipid monolayer

Summary for 9Y48

• Entry DOI: 10.2210/pdb9y48/pdb
• EMDB information: 72474
• Descriptor: Lethal(2) giant larvae protein homolog SRO7, SODIUM ION (2 entities in total)
• Functional Keywords: exocytosis, membrane trafficking, vesicle tethering
• Biological source: Saccharomyces cerevisiae (brewer's yeast)
• Total number of polymer chains: 1
• Total formula weight: 114662.41

Authors

Baker, R.W.,Strauss, J.D. (deposition date: 2025-09-02, release date: 2025-10-29, Last modification date: 2025-11-19)

Primary citation

Skrajna, A.,Lenger, C.,Robinson, E.,Cannon, K.,Sarsam, R.,Ouellette, R.G.,Abotsi, A.M.,Brennwald, P.,McGinty, R.K.,Strauss, J.D.,Baker, R.W.
Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM.
J.Struct.Biol., 217:108253-108253, 2025

PubMed Abstract: 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 (∼30 nm). We determined three structures ranging from 2.4 to 3.0 Å resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 Å 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.

PubMed: 41083086
DOI: 10.1016/j.jsb.2025.108253

Experimental method

ELECTRON MICROSCOPY (3.1 Å)