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TitleGenetically encoded multimeric tags for subcellular protein localization in cryo-EM.
Journal, issue, pagesNat Methods, Vol. 20, Issue 12, Page 1900-1908, Year 2023
Publish dateNov 6, 2023
AuthorsHerman 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 AbstractCryo-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 linksNat Methods / PubMed:37932397 / PubMed Central
MethodsEM (subtomogram averaging) / EM (tomography)
Resolution13.7 - 31.3 Å
Structure data

EMDB-16303: In situ subtomogram average of GEM2 particles in human cells
Method: EM (subtomogram averaging) / Resolution: 31.3 Å

EMDB-18194: Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells
Method: EM (tomography) / Resolution: 13.7 Å

Source
  • Synechococcus elongatus PCC 7942 = FACHB-805 (bacteria)
  • Homo sapiens (human)

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