Bacterial and archaeal cells are surrounded by a cell membrane and cell wall components such as the peptidoglycan, outer membrane, surface proteins (S-layer), or other specialised layers in various combinations. We are interested in the structure, interaction and function of protein complexes located in the cell envelope and in the periplasm of microorganisms. Protein complexes of interest are pore-forming outer membrane proteins of mycobacteria and other functional protein assemblies of selected archaea and bacteria.
We study the location and 3D structure of protein complexes in situ by means of cryo-electron tomography of intact (vitrified) cells using the whole armamentarium of specimen thinning, optimized imaging and data reconstruction to enhance the resolution and to extract the information of small structural details. To make use of the full information contained in tomograms it is necessary to correct for the effects of the contrast transfer function (CTF) in projections prior to 3D reconstruction. We introduced an acquisition scheme that allows us to determine the CTF of individual projections of a tomographic data set and to recover structural details of membrane protein complexes in situ to a resolution of better than 2 nm after correction and subtomogram averaging. This approach promises to reveal the structure and interactions of macromolecular assemblies in the cell envelope and in native microbial cells to a unprecedented resolution.
Engelhardt H.: Visualizing the bacterial cell surface: an overview. Meth. Mol. Biol. 966: 15-35, 2013
Eibauer M., Hoffmann C., Plitzko J.M., Baumeister W., Nickell S. and Engelhardt H.: Uraveling the structure of membrane proteins in situ by transfer function corrected cryo-electron tomography. J. Struct. Biol. 180: 488-496, 2012
Strunk T., Hamacher K., Hoffgaard F., Engelhardt H., Zillig M.D., Faist K., Wenzel W. and Pfeifer F.: Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo. Mol. Microbiol. 81: 56-68, 2011
Niederweis M., Danilchanka O., Huff J., Hoffmann C. and Engelhardt H.: Mycobacterial outer membranes: in search of proteins. Trends Microbiol. 18: 109-116, 2010
Hoffmann C., Leis A., Niederweis M., Plitzko J.M. and Engelhardt H.: Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl. Acad. Sci. USA 105: 3963-3967, 2008
Engelhardt H.: Are S-layers exoskeletons? The basic function of surface protein layers revisited. J. Struct. Biol. 160: 115-124, 2007
Engelhardt H.: Mechanism of osmoprotection by archaeal S-layers: A theoretical study. J. Struct. Biol. 160: 190-199, 2007