Recently, Associate Professor Shen Qingtao's research group at the School of Life Sciences, Southern University of Science and Technology, tackled the challenge of flexible assembly of biological macromolecules. They developed a reconstruction method based on cryo-electron microscopy single-particle technology that directly assigns Euler angles to particles, elucidating the structure of the planar helical polymer of the endosomal sorting complex required for transport (ESCRT-III) in eukaryotic cells. The research findings were published in the Proceedings of the National Academy of Sciences of the United States of America.
ESCRT is an evolutionarily conserved class of biological macromolecular machinery found widely in archaea, bacteria, and eukaryotes. It is responsible for membrane remodeling processes in eukaryotic cells that deviate from the cytoplasmic direction, such as cell division, nuclear membrane remodeling, plasma membrane repair, and neuronal pruning. Dysfunction of ESCRT machinery can lead to the occurrence of diseases like cancer and neurodegenerative disorders.
The ESCRT molecular machinery consists of five subcomplexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. Among these, ESCRT-III is crucial for the most important membrane deformation and membrane scission processes in membrane remodeling. Previously, Shen Qingtao's team observed the planar helical polymer form of ESCRT-III using cryo-electron microscopy. However, the characteristics of the ESCRT-III planar helical polymer, such as thin diameter, high curvature, and severe preferred orientation, posed significant challenges to its three-dimensional structural analysis, severely limiting the understanding of the membrane remodeling mechanism mediated by ESCRT-III.
Based on the central section theorem, the key step in 3D reconstruction lies in calculating the precise Euler angles for each particle. Currently, Euler angles are obtained through particle-template matching, which easily falls into local optima traps, leading to reconstruction errors.
In this study, researchers collected tilted sample data, developed graphic software to depict the planar helical polymer of ESCRT-III, fully utilized geometric constraints between adjacent particles, calculated the initial Euler angles of particles through mathematical computation, and developed a reconstruction method that directly assigns Euler angles to particles. This method successfully resolved the three-dimensional structures of different conformations of ESCRT-III, outlining the assembly form and mechanism of ESCRT-III on the membrane.
This work provides an initial conformation for the transition of ESCRT-III polymer from planar helix to circular crown structure, offering a new direction for studying membrane remodeling mediated by ESCRT-III. The reconstruction method proposed in this study effectively avoids the problem of local optima in single-particle reconstruction using cryo-electron microscopy and provides a new strategy for analyzing the structures of large molecular complexes with severe preferred orientation on membrane systems like liposomes and organelles.
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