Plunge freezing tweezers with ultrasonic excitation for reducing preferred particle orientation in cryo-EM
- 1Ecole Polytechnique Federale de Lausanne (EPFL), Laboratory of Molecular Nanodynamics, CH-1015 Lausanne, Switzerland
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 [A].
Related
- Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers
- Simulating Bead-Tethered Biomolecules: Force-Probe Coupled Steered Molecular Dynamics (fp-SMD) in Optical Tweezers Force Spectroscopy
- COPAL: An ensemble of protein-ligand co-folding models enriches preferred ligand predictions for the LuxR-family of quorum sensing receptors
- Freezing-induced solid-liquid microphase separation for self-assembly of functional nucleic acid nanoparticles
- Micro-Flow Imaging and Flow Virometry as Preferred Methods to Monitor Aggregate Formation in the Submicron and Subvisible Size Range for Two Early-Phase Vaccine Candidates
BibTeX
@misc{wenz2026plunge,
title = {{Plunge freezing tweezers with ultrasonic excitation for reducing preferred particle orientation in cryo-EM}},
author = {J. Wenz and M. Flor and C. R. Krueger and M. Drabbels and U. J. Lorenz},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.29.755300},
url = {https://www.biorxiv.org/content/10.64898/2026.09.29.755300v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Plunge freezing tweezers with ultrasonic excitation for reducing preferred particle orientation in cryo-EM
AU - J. Wenz
AU - M. Flor
AU - C. R. Krueger
AU - M. Drabbels
AU - U. J. Lorenz
PY - 2026
DA - 2026/09/29
PB - bioRxiv
DO - 10.64898/2026.09.29.755300
UR - https://www.biorxiv.org/content/10.64898/2026.09.29.755300v1
AB - 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 [A].
ER -