A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy.
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.
School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, University Walk BS8 1TD, United Kingdom.
Martin
M
Cheung
Naoko
N
Kajimura
Fumiaki
F
Makino
Masamichi
M
Ashihara
Tomoko
T
Miyata
Takayuki
T
Kato
Keiichi
K
Namba
Ariel J
AJ
Blocker
D002244:Carbon / Q000737:chemistry*; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D046911:Macromolecular Substances / Q000737:chemistry*; D058989:Vitrification
D046911:Macromolecular Substances; D002244:Carbon