Three-dimensional imaging of in situ specimens with low-dose electron tomography to analyze protein conformation.
We describe a novel three-dimensional (3-D) imaging tool for analysis of protein conformation of in situ samples. Sidec (Sidec Technologies AB, Stockholm, Sweden) electron tomography (SET) uses low-dose electron tomography and a refinement algorithm to reconstruct individual proteins and macromolecular complexes. The approach has successfully reconstructed therapeutic proteins in solution. In this study, we investigate the use of SET to visualize ion channels in cells and tissue samples. SET successfully resolved the volume and structural features of the target complex, showing that it was a tetrameric channel with a central pore. The technology could distinguish and provide 3-D images of the intra- and extracellular domains in the ion channel. In addition, SET was able to show that the channel associates in the form of a tetramer with the four subunits preorganized into dimers. While additional studies using smaller antibody markers are needed to resolve the subunit assembly further, this study demonstrates that SET is a valuable tool for visualization of in situ specimens and can provide important information on the subunit assembly of these macromolecular complexes, and thereby aid in the screening assay process in drug development.
Banyay
Martina
M
Sidec Technologies AB, Fogdevreten 2A, SE-17177 Stockholm, Sweden.
Gilstring
Fredrik
F
Hauzenberger
Elenor
E
Ofverstedt
Lars-Göran
LG
Eriksson
Anders B
AB
Krupp
Johannes J
JJ
Larsson
Olof
O
D000818:Animals; D004583:Electrons; D007091:Image Processing, Computer-Assisted / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007473:Ion Channels / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D014054:Tomography / Q000379:methods*
D007473:Ion Channels