- EMDB-16303: In situ subtomogram average of GEM2 particles in human cells -
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Basic information
Entry
Database: EMDB / ID: EMD-16303
Title
In situ subtomogram average of GEM2 particles in human cells
Map data
FSC-weighted non-sharpened map
Sample
Cell: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
Protein or peptide: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
Keywords
Encapsulin / Protein Engineering / CYTOSOLIC PROTEIN
Function / homology
Type 2A encapsulin shell protein SrpI-like / : / Type 2A encapsulin shell protein SrpI-like / encapsulin nanocompartment / Type 2A encapsulin shell protein SrpI
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND
664726
Germany
H2020 Marie Curie Actions of the European Commission
101028297
European Union
Human Frontier Science Program (HFSP)
CDA00045/2019
France
Other private
Biomedicum Helsinki Foundation
Other private
Orion Foundation
Citation
Journal: Nat Methods / Year: 2023 Title: Genetically encoded multimeric tags for subcellular protein localization in cryo-EM. 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 / 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.
Entire : Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...
Entire
Name: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
Components
Cell: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
Protein or peptide: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
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Supramolecule #1: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...
Supramolecule
Name: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2 type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Macromolecule #1: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...
Macromolecule
Name: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2 type: protein_or_peptide / ID: 1 Details: Fusion of Synechococcus elongatus Srp1 with Halo-tag and the FKBP-rapamycin-binding (FRB) domain of mTOR Enantiomer: LEVO
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