Plunge freezing tweezers with ultrasonic excitation for reducing preferred particle orientation in cryo-EM

Abstract Preferred particle orientation, which results from the adsorption of proteins to the air-water interface, is a frequently encountered issue in single-particle cryo-electron microscopy (cryo-EM). Particles then present only a limited number of views, which are oversampled during data acquisition at the expense of throughput, while other views may be missing altogether. In severe cases, a high-resolution reconstruction becomes unattainable. We have recently established that ultrasonic excitation of the sample during vitrification improves the angular distribution by detaching particles from the air-water interface and reshuffling their orientations. Here, we describe an implementation of this principle for a plunge freezing apparatus, in which the ultrasonic transducer is integrated into the head of the pair of tweezers holding the sample, and show that this design is able to reduce preferred orientation. We demonstrate this for hemagglutinin, the canonical example of preferred orientation, and show that the fraction of side and tilted views increases with the excitation amplitude. This allows us to obtain a complete reconstruction of the trimer at 2.5 Å.