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Volume 5
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10.3390/biophysica5030030
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Article

Optimizing Single-Particle Analysis Workflow: Comparative Analysis of the Symmetry Parameter and Particle Quantity upon Reconstruction of the Molecular Complex

by
Myeong Seon Jeong
1,2,†,
Han-ul Kim
1,3,†,
Mi Young An
1,
Yoon Ho Park
1,
Sun Hee Park
4,5,
Sang J. Chung
4,5,
Yoon-Sun Yi
2,
Sangmi Jun
2,
Young Kwan Kim
3 and
Hyun Suk Jung
1,3,*
1
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
2
Center for Bio-Imaging Translational Research, Korea Basic Science Institute, Cheongju 28119, Republic of Korea
3
Kangwon Center for Systems Imaging, Chuncheon 24341, Republic of Korea
4
School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea
5
AbTis Co., Ltd., Suwon 16648, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biophysica 2025, 5(3), 30; https://doi.org/10.3390/biophysica5030030
Submission received: 20 June 2025 / Revised: 5 July 2025 / Accepted: 15 July 2025 / Published: 22 July 2025
(This article belongs to the Special Issue Investigations into Protein Structure)
Versions Notes

Abstract

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing.
Keywords: pyruvate dehydrogenase complex; molecular complex; cryo-electron microscopy; image processing; single-particle analysis pyruvate dehydrogenase complex; molecular complex; cryo-electron microscopy; image processing; single-particle analysis

Share and Cite

MDPI and ACS Style

Jeong, M.S.; Kim, H.-u.; An, M.Y.; Park, Y.H.; Park, S.H.; Chung, S.J.; Yi, Y.-S.; Jun, S.; Kim, Y.K.; Jung, H.S. Optimizing Single-Particle Analysis Workflow: Comparative Analysis of the Symmetry Parameter and Particle Quantity upon Reconstruction of the Molecular Complex. Biophysica 2025, 5, 30. https://doi.org/10.3390/biophysica5030030

AMA Style

Jeong MS, Kim H-u, An MY, Park YH, Park SH, Chung SJ, Yi Y-S, Jun S, Kim YK, Jung HS. Optimizing Single-Particle Analysis Workflow: Comparative Analysis of the Symmetry Parameter and Particle Quantity upon Reconstruction of the Molecular Complex. Biophysica. 2025; 5(3):30. https://doi.org/10.3390/biophysica5030030

Chicago/Turabian Style

Jeong, Myeong Seon, Han-ul Kim, Mi Young An, Yoon Ho Park, Sun Hee Park, Sang J. Chung, Yoon-Sun Yi, Sangmi Jun, Young Kwan Kim, and Hyun Suk Jung. 2025. "Optimizing Single-Particle Analysis Workflow: Comparative Analysis of the Symmetry Parameter and Particle Quantity upon Reconstruction of the Molecular Complex" Biophysica 5, no. 3: 30. https://doi.org/10.3390/biophysica5030030

APA Style

Jeong, M. S., Kim, H.-u., An, M. Y., Park, Y. H., Park, S. H., Chung, S. J., Yi, Y.-S., Jun, S., Kim, Y. K., & Jung, H. S. (2025). Optimizing Single-Particle Analysis Workflow: Comparative Analysis of the Symmetry Parameter and Particle Quantity upon Reconstruction of the Molecular Complex. Biophysica, 5(3), 30. https://doi.org/10.3390/biophysica5030030

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