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| Title | Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å. |
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| Journal, issue, pages | Sci Rep, Vol. 14, Issue 1, Page 14079, Year 2024 |
| Publish date | Jun 18, 2024 |
 Authors | Hsin-Hung Lin / Chun-Hsiung Wang / Shih-Hsin Huang / Sung-Yao Lin / Takayuki Kato / Keiichi Namba / Naoki Hosogi / Chihong Song / Kazuyoshi Murata / Ching-Hsuan Yen / Tsui-Ling Hsu / Chi-Huey Wong / Yi-Min Wu / I-Ping Tu / Wei-Hau Chang /   |
| PubMed Abstract | While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small ...While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features. |
 External links | Sci Rep / PubMed:38890341 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 5.69 - 8.45 Å |
| Structure data |  EMDB-38153: Immunoglobulin (IgG) reconstruction using phase plate single-particle cryo-EM  EMDB-38154: Fab domain of Immunoglobulin (IgG) reconstruction using phase plate single-particle cryo-EM  EMDB-38155: Fab domain of Immunoglobulin (IgG) reconstruction using conventional single-particle cryo-EM |
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Mus musculus (house mouse)