A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy

Abstract

Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.

Fig.1

HBB samples applied to glow discharged grid and grids pretreated with various detergents. (A) Glow discharged grid – The majority of HBBs adsorbed to the carbon support film. (B) DDM pretreated grid – The adsorption of HBBs onto carbon surface was significantly reduced compared with glow discharged grids. However, DDM pretreatment moderately aggravated the aggregation of HBBs. (C) CTAB pretreated grid – A high concentration of well dispersed HBBs were seen, with next to no attachment to carbon. (D) LDAO pretreated grid – Grids treated with LDAO showed a remarkable reduction in particle adsorption to the carbon, but fewer particles were also found the holes themselves. Scale bars represent 200 nm.

Fig.2

Surface wetting by detergent treatment. (A) Diagram illustrating Young’s relationship for a drop sitting on a surface, which describes the equilibrium established between the solid–liquid (S/L), liquid–vapour (L/V) and solid–vapour (S/V) inter-facial energies (γ). (B) Measurements of the contact angle, θ, created by a drop of water sitting on non-glow discharged (top), LDAO washed (middle) and glow discharged grids (bottom).