Oral Presentation 2014 International Biophysics Congress

CryoEM image analysis of macromolecular motor assemblies (#3)

Keiichi Namba 1
  1. Osaka University, Suita, Osaka, Japan

High resolution structures of macromolecular assemblies and cellular architectures are essential for understanding the mechanisms of biological functions because they are all determined by their structures and dynamics at atomic level. X-ray crystallography and NMR are powerful tools but the difficulty in crystallization and the size limit of target molecules pause severe limitations on the former and the latter, respectively. Filamentous protein complexes are particularly difficult target because the length is not uniform. Electron cryomicroscopy (cryoEM) and image analysis is a powerful tool because there is no size limitation and it requires a very small amount of sample. The achievable resolution is, however, limited by intrinsically low signal to noise ratio (S/N) of cryoEM images due to extremely low electron dose to avoid radiation damage. Therefore, tens of thousands of images have to be collected, classified, aligned and averaged to reconstruct high-resolution 3D images. However, accurate image alignment is itself difficult due to the low S/N of images. We developed techniques to improve the image S/N and speed of data collection by use of an energy filter and CCD camera with the liquid helium-cooled specimen stage on an electron microscope operated at 200 - 300 kV. We have gained nearly 5 times higher S/N by controlling ice thickness and raising the specimen temperature from 4 K to 50 K. The undesirable charging effect has been minimized at 50 K, allowing all the images to be used for analysis, compared to a few % at 4 K. Single particle image analysis now allows the high-resolution structural analysis of filamentous protein complexes to be completed within a week, and the achievable resolution is beyond 4 Å. I will describe the structures of various filamentous protein complexes and discuss the potential of the method for future life sciences.