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Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon
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Integrating multitemperature sample preparation with particle rebalancing addresses preferred orientation
Xin Su1,2,3,4, Liuyan Yang2,3,4, Caiying Zhang2,3,4
1State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
PNAS Nexus
|August 8, 2026
Summary
Researchers developed a new method to improve single-particle cryo-electron microscopy (cryo-EM) by adjusting sample preparation temperature. This technique enhances particle orientation and boosts reconstruction quality for better structural analysis.
Area of Science:
- Structural Biology
- Biophysics
- Cryo-Electron Microscopy
Background:
- Single-particle cryo-electron microscopy (cryo-EM) is a powerful technique for determining the three-dimensional structure of biological macromolecules.
- A major challenge in cryo-EM is preferred particle orientation, where sample particles preferentially align in a specific direction, leading to reconstruction artifacts and reduced resolution.
- Existing methods to overcome preferred orientation, such as detergents, grid modifications, tilted data collection, and algorithmic corrections, are often complex, time-consuming, and specific to each sample.
Purpose of the Study:
- To develop a facile and effective method to modulate particle orientation in cryo-EM.
- To improve the quality of cryo-EM reconstructions by addressing the issue of preferred orientation.
- To provide a broadly applicable strategy for enhancing cryo-EM data quality.
Main Methods:
- Investigated the effect of varying cryo-EM sample preparation temperatures on particle orientation.
- Collected thermophilic 70S ribosome datasets at different temperatures.
- Applied a three-dimensional (3D) reconstruction-based particle rebalancing strategy to merged datasets.
Main Results:
- Achieved improved cryo-EM reconstruction quality, evidenced by smoother directional Fourier shell correlation (FSC) curves.
- Observed higher 3D FSC sphericity and a narrower anisotropy transition zone in Fourier shell occupancy.
- Successfully determined the high-resolution structure of γ-glutamylmethylamide synthetase, a complex known for severe preferred orientation issues.
Conclusions:
- Integrating high-temperature sample preparation with particle rebalancing is an effective approach to mitigate preferred orientation in cryo-EM.
- This method offers a simpler and more efficient alternative to existing techniques for improving cryo-EM reconstructions.
- The proposed strategy demonstrates broad potential applicability across various single-particle cryo-EM studies.
