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Published on: November 10, 2023
Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM
Aleksandra Skrajna1, Clara Lenger2, Emily Robinson2
1Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, Chapel Hill, NC, USA; UNC Lineberger Comprehensive Cancer Center, UNC Chapel Hill School of Medicine, Chapel Hill, NC, USA.
This study introduces a straightforward method using nickel nitrilotriacetic acid (Ni-NTA) lipid affinity grids to improve single particle cryo-electron microscopy (cryo-EM) grid preparation. These grids concentrate proteins, protect them from air-water interface damage, and enable high-resolution structure determination.
Area of Science:
- Structural Biology
- Biophysics
- Cryo-Electron Microscopy (Cryo-EM)
Background:
- Grid preparation is a critical bottleneck in achieving high-resolution structures via single particle cryo-EM.
- Particle behavior at the air-water interface (AWI) can lead to denaturation, aggregation, or preferred orientation, compromising data quality.
- Traditional methods may yield insufficient particle quantities, necessitating solid supports for sample concentration.
Purpose of the Study:
- To develop and systematically analyze a straightforward preparation method for Ni-NTA lipid monolayers as affinity grids for high-resolution single particle cryo-EM.
- To evaluate the effectiveness of these affinity grids in concentrating samples and mitigating AWI-induced artifacts.
- To demonstrate the utility of this method for determining high-resolution structures of various biological macromolecules.
Main Methods:
- Preparation of Ni-NTA lipid monolayers on grid supports.
- Application of the affinity grids to biological samples, allowing selective binding of poly-histidine tagged proteins.
- Systematic analysis of particle distribution, ice thickness, and resulting cryo-EM structure resolution.
Main Results:
- Lipid affinity grids effectively concentrate particles away from the AWI in thin ice (approximately 30 nm).
- Three structures were determined with resolutions ranging from 2.4 to 3.0 Å, confirming the method's suitability for high-resolution imaging.
- A 3.1 Å resolution structure of a sub-100 kDa protein, determined without symmetry, highlights the method's broad applicability.
Conclusions:
- Ni-NTA lipid monolayers provide a simple and effective approach for cryo-EM grid preparation, overcoming common challenges.
- This method enhances particle yield, preserves sample integrity against AWI damage, and facilitates high-resolution structure determination.
- Lipid affinity grids are a versatile and extendable tool for a wide range of biological systems in cryo-EM studies.
