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Updated: May 5, 2026
High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis
Published on: October 15, 2019
An efficient clear-native PAGE-based workflow for cryo-electron microscopy sample preparation of large protein
Zitong Yang1,2, Shinsa Kameo1,2, Soichiro Seki3
1Institute of Low Temperature Science, Hokkaido University, N19 W8 Kita-Ku, Sapporo, 060-0819, Japan.
Background:
Cryo-electron microscopy (cryo-EM) has revolutionized protein research by enabling high-resolution structural analysis. However, preparing ultra-large protein complexes (e.g., > 700 kDa) for cryo-EM remains challenging, as it requires preserving both structural integrity and the native state. Conventional isolation methods, such as sucrose density gradient centrifugation, require large sample volumes and provide limited separation resolution. In contrast, native PAGE offers higher resolution; however, no established method exists for extracting protein complexes from gels followed by further purification to achieve high purity. Consequently, no standardized native PAGE-based protocol for cryo-EM sample preparation avoids multiple purification steps. Hence, we aimed to develop a rapid and efficient cryo-EM protein sample preparation method using electroelution with an optimized buffer system that preserves complex integrity to recover target protein complexes after sodium deoxycholate (DOC)-based clear-native PAGE (CN-PAGE).
Results:
We developed an agarose-acrylamide composite gel, which is simpler to prepare and mechanically more robust than conventional linear-gradient acrylamide gels commonly used for CN-PAGE, facilitating precise band excision for efficient electroelution. Cryo-EM structural analysis of the photosystem I-light-harvesting complex I (PSI-LHCI) supercomplex from Arabidopsis thaliana achieved high resolution (2.18 Å) after electroelution from this gel, requiring only buffer exchange by ultrafiltration to remove DOC before grid preparation, without additional chromatographic purification. This finding suggests that DOC may be the main inhibitor of successful grid preparation.
Conclusion:
Our results demonstrate the potential of this method for isolating large protein complexes from small sample volumes for cryo-EM structural analysis. This approach may expand the scope of cryo-EM targets to include some challenging systems previously hindered by purification difficulties.
