A Surfactant Cocktail Overcomes Air-Water Interface Artifacts in Single-Particle CryoEM

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Single-particle cryo-electron microscopy (cryoEM) determines biomacromolecular structures to near-atomic resolution.
• Vitrification-induced air-water interface (AWI) interactions cause preferred orientation and denaturation, hindering 3D reconstruction.
• Current surfactant strategies for mitigating AWI effects require laborious, sample-specific screening.

Purpose of the Study:

• To develop a universal surfactant solution to overcome AWI-induced challenges in cryoEM sample preparation.
• To reduce orientation bias and denaturation for improved 3D reconstructions.
• To minimize the need for extensive surfactant screening for diverse protein samples.

Main Methods:

• Development of SurfACT, a cocktail of surfactants with varied physicochemical properties.
• Application of SurfACT to four challenging protein samples: hemagglutinin (HA), molybdenum-iron protein (MoFeP), and aldolase.
• Analysis of particle distribution, orientation, and 3D map quality using cryoEM and cryogenic electron tomography.

Main Results:

• SurfACT significantly reduced AWI-induced orientation bias and denaturation in all tested proteins.
• Improved particle distribution, with SurfACT redistributing particles from the AWI into the bulk ice.
• Enhanced 3D reconstruction quality and map completeness for samples previously limited by AWI effects.

Conclusions:

• SurfACT is a versatile additive that effectively mitigates AWI-related issues in cryoEM sample preparation.
• This approach expands the range of suitable samples for cryoEM and streamlines the structure determination process.
• SurfACT minimizes the need for laborious, sample-specific surfactant screening, accelerating structural studies.