Tecnai Polara G2 TEM electron microscope
Cryo-electron microscopy (EM), together with associated image processing techniques, has become an established method of structural analysis of large macromolecular complexes.
It has become possible to reconstruct and visualize three-dimensional density distributions of objects ranging from individual proteins, to protein-RNA complexes, such as ribosomes, to highly symmetric structures of viruses. Although in terms of the best resolution achieved single-particle cryo-EM cannot yet match X-ray crystallography, there are many advantages of this technique. Thanks to the rapid freezing of the specimen, molecules are captured in their native, aqueous environment, and their native structure is preserved. Therefore, it is possible to study conformational changes and to examine dynamical effects of different functional states. Intermediate-resolution maps provide a wealth of structural information including the determination of the overall structure and the localization of major domains. The calculation of difference maps can reveal the location of smaller molecules bound to larger complexes. Docking of X-ray structures of molecules into EM density maps can reveal the arrangement of known molecules within the EM envelope. Finally, EM is unique in its ability to reveal the conformational variability of macromolecular complexes.
Cryo-EM is an advanced technique that requires successful purification of proteins, imaging to high resolution under stringent vacuum conditions in the electron microscope and, last but not least, advanced computational techniques to build the final model of the structure. This challenging work has to bring together researchers from fields ranging from biochemistry to computational biology.
The backbone of the EM facility is Tecnai Polara G2 300 kV liquid helium cooled electron microscope from FEI company equipped with Tietz 4k x 4k CCD camera and a field emission gun (FEG) electron source. In addition, the microscope is capable of tilting the specimen computer controlled stage from -70 to +70 degrees to collect, in an automated mode, electron tomographic data. The Center also provides access to a JEOL 1200EX 100kV electron microscope equipped with cryo stage located in the Pathology Department of the School. Cryo-EM specimen preparation is conducted using a robotic cryo-preparation unit Vitrobot. The cryo-EM structure determination expertise is provided by Dr. Penczek, who develops, together with collaborators from the Baylor College of Medicine, Houston, and Lawrence Berkeley Laboratory, Berkeley, CA, a novel single particle software package SPARXhttp://macro-em.org/sparxwiki/.