Wang, ChangChangWangKhosrozadeh, AminAminKhosrozadehIacovache, IoanIoanIacovacheZuber, BenoîtBenoîtZuber0000-0001-7725-55792025-09-112025-09-112025-09-03https://boris-portal.unibe.ch/handle/20.500.12422/218277Cryo-electron tomography (cryoET) provides 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. However, locating target proteins in live cells remains a significant challenge. Conventional labeling methods, such as fluorescent protein tagging and immunogold labeling, are unsuitable for small structures in vitrified samples at molecular resolution. Directly linking large, visually identifiable proteins to target proteins may alter their structure, localization, and function. To overcome this, we employed a rapamycin-induced oligomer formation system involving two tags, FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB), which bind in the presence of rapamycin. FKBP is linked to the target protein, while FRB is linked to ferritin, a large (10-12 nm) iron-binding complex that creates strong contrast in cryoET. Upon adding rapamycin to the cell medium, the iron-loaded ferritin accurately marks the target protein location. As in situ cryoET with subtomogram averaging advances, our method addresses the persistent challenge of locating target proteins in live cells.enCEMOVIScryo-electron microscopycryo-electron tomographycryoFIB millinglabeling600 - Technology::610 - Medicine & healtharticle10.48620/912774092536410.1016/j.str.2025.08.013