Cryo-Electron Microscopy Core
Director: Peijun Zhang, PhD
The CryoEM Core applies this technique to 1) characterize the structural features of HIV and host protein complexes, by negative staining, to determine their suitability for further structural study; 2) determine the structures of complexes/assemblies deemed suitable; and 3) develop methods for single particle cryoEM and cryoET to analyze structural heterogeneity. The Core utilizes established protocols to structurally characterize individual complexes and assemblies with diverse characteristics. The protocols include Iterative Helical Real-Space Reconstruction (IHRSR); single particle analysis of large multi-protein complexes using EMAN and Frealign software packages, and cryo-ET reconstruction and sub-volume classification and averaging methods using IMOD.
The PCHPI makes use of the cryo-EM facility in the Department of Structural Biology in the basement of the BST3. The facility houses state-of-the-art electron microscopy instruments and has full capability to carry out high resolution three-dimensional structural analysis of proteins, protein complexes, phages and viruses, macromolecular assemblies, cellular organelles, and bacterial cells. Three electron microscopes are available: a Polara G2 electron microscope operating at 300kv and a Tecnai F20 electron microscope operating at 200kv, both equipped with high-resolution 4kx4k CCD cameras and capable of low-dose cryo-EM and electron tomographic studies. A Tecnai T12spirit, operating at 120kv, handles room temperature specimens and has features for automatic tuning and alignment. The facility is also equipped with a FEI vitrobot for frozen-hydrated specimen preparation and other ancillaries for cryoEM.