“Membrane Protein Structure Analysis by Electron Microscopy”
Henning Stahlberg, Assistant Professor – Department of Molecular and Cellular Biology, University of CA at Davis
Membrane proteins are central to health and disease. The determination of their structure faces challenges in the membrane protein production, purification and 3D crystallization. Cryo-Electron Microscopy (cryo-EM) of two-dimensional membrane crystals can provide an attractive alternative structure determination method, since the success-rate for growing 2D crystals is higher than growing 3D crystals, and since smaller protein quantities are needed. However, electron microscopy analysis is usually limited to lower resolutions, especially in the direction perpendicular to the membrane. This is due to the so-called missing Fourier-cone effect, but also due to a beam-induced apparent specimen movement, which can limit the resolution in images of tilted samples. And the electron microscopy data collection and computer data analysis are labor intensive, and therefore slow.
To address the resolution loss due to beam-induced sample movement, we test the usability of Scanning Transmission Electron Microscopy for 2D crystal samples. First data will be presented documenting that STEM imaging of frozen hydrated samples is not suffering from the beam-induced apparent specimen drift. STEM imaging may therefore represent a possibility to avoid the major resolution-limiting factor in cryo-EM imaging. However, STEM imaging of beam-sensitive biological samples will require the adaptation of conventional cryo-EM imaging methods.
The data processing of 2D crystal data is usually done with the MRC program suite (Crowther et al., J. Struct. Biol., 116(1):9-16 (1996). Based on the MRC software, we have developed a new software system called 2dx, which will be presented. 2dx allows the user-friendly, and optionally fully automatic processing of 2D crystal images, and therefore can eliminate the speed bottleneck in the data analysis.
Finally, progress on the structural analysis of ion channels by single particle cryo-EM and by electron crystallography of 2D crystals will be presented.