“Recent Advances in Cryoelectron Tomography of Frozen Hydrated Samples”
Grant Jensen – CalTech
A new helium-cooled, 300kV, FEG, G2 Polara FEI TEM was installed at Caltech in the fall of 2003 with several technological advances which we have now tested and exploited to image frozen-hydrated proteins, viruses, and bacteria. Our initial results will be reported. Firstly, the new “flip-flop” cryo-rotation stage has allowed us to routinely record dual-axis tilt series, significantly improving the point-spread-function in our tomograms. Secondly, this cartridge-based system has allowed us to record tilt series without extra tracking or focusing images through UCSF’s “predictive” tomography package, so that a 70 image, single-axis, 2k by 2k tilt series takes only about 30 minutes. Disappointingly, helium-cooling did not prove advantageous, and in fact is generally disadvantageous as compared to nitrogen-cooling for electron cryotomography. Tomographic reconstructions of HIV-1 virus-like particles will be presented with important implications concerning the structure and assembly of the capsid shell. Finally, tomographic reconstructions of individual protein complexes will be shown which clearly reveal the relative positions of 80kDa protein domains, establishing electron cryotomography as a powerful new structural technique for understanding the conformational dynamics of macromolecular machines.