+Search query
-Structure paper
Title | Genetically encoded multimeric tags for subcellular protein localization in cryo-EM. |
---|---|
Journal, issue, pages | Nat Methods, Vol. 20, Issue 12, Page 1900-1908, Year 2023 |
Publish date | Nov 6, 2023 |
Authors | Herman K H Fung / Yuki Hayashi / Veijo T Salo / Anastasiia Babenko / Ievgeniia Zagoriy / Andreas Brunner / Jan Ellenberg / Christoph W Müller / Sara Cuylen-Haering / Julia Mahamid / |
PubMed Abstract | Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest ...Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function. |
External links | Nat Methods / PubMed:37932397 / PubMed Central |
Methods | EM (subtomogram averaging) / EM (tomography) |
Resolution | 13.7 - 31.3 Å |
Structure data | EMDB-16303: In situ subtomogram average of GEM2 particles in human cells EMDB-18194: Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells |
Source |
|