Genetically Encoded FerriTag as a Specific Label for Cryo-Electron Tomography

Cryo-electron tomography (cryoET) is an important imaging technique that can provide 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. Recently, combining cryoET with subtomogram averaging has pushed the resolution to 3-4 A. However, one main challenge for cryoET is locating target proteins in live cells. Conventional methods such as fluorescent protein tagging and immunogold labeling are not entirely suitable to label small structures in live cells with molecular resolution in vitrified samples. If large proteins, which can be visually identified in cryoET, are directly linked to the target protein, the large tag may alter the target protein structure, localization and function. To address this challenge, we used the rapamycin-induced oligomer formation system, which involves two tags (FKBP and FRB) that can bind together within rapamycin. In our system, the FKBP tag is linked to target protein and the FRB tag is linked to a large protein to create a marker. We chose ferritin as the marker protein because it is a large complex (10-12 nm) and can bind iron to create strong contrast in cryoET. After adding rapamycin to the cell medium, the iron-loaded ferritin accurately indicates the location of the target protein. Recently, in-situ cryoET with subtomogram averaging has been rapidly developing. However, it is still challenging to locate target proteins in live cells, and this method provides a much-needed solution.