Visualization of proteins in intact cells with a clonable tag for electron microscopy. Diestra, E., Fontana, J., Guichard, P., Marco, S., & Risco, C. Journal of Structural Biology, 165(3):157--168, March, 2009. ![Visualization of proteins in intact cells with a clonable tag for electron microscopy [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.
@article{diestra_visualization_2009,
title = {Visualization of proteins in intact cells with a clonable tag for electron microscopy},
volume = {165},
issn = {1047-8477},
url = {http://www.sciencedirect.com/science/article/pii/S104784770800289X},
doi = {10.1016/j.jsb.2008.11.009},
abstract = {Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.},
number = {3},
urldate = {2014-05-19TZ},
journal = {Journal of Structural Biology},
author = {Diestra, Elia and Fontana, Juan and Guichard, Paul and Marco, Sergio and Risco, Cristina},
month = mar,
year = {2009},
keywords = {AmiC, Clonable gold tags, E. coli proteins, Electron microscopy, Electron tomography, Maltose binding protein, RecA},
pages = {157--168}
}
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The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. 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