Overcoming Preferred Orientation in Cryo-EM With Ultrasonic Excitation During Vitrification
Williams, H. M.; Curtis, W. A.; Haubner, M.; Hruby, J.; Drabbels, M.; Lorenz, U. J.
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Preferred particle orientation remains a frequently encountered issue in cryo-electron microscopy that arises when proteins adsorb to the air-water interface in only a limited number of orientations. This can significantly increase the data acquisition time required to reach a desired resolution or even make it impossible to obtain a reconstruction altogether. Here, we show that preferred orientation can be overcome by continuously exciting the sample with ultrasonic waves during vitrification. Our experiments suggest that mechanical oscillations induced in the sample support continuously shake proteins loose form the air-water interface, thereby scrambling their orientations. The simple, physical nature of this mechanism should make it applicable to a wide range of proteins. Since our method can be easily implemented in existing vitrification devices, it should find widespread adoption.
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