SCIENTIFIC CORES
Bacterial Protein Expression Core
Biochemistry Core Cell Biology and Imaging Core Computation Core EM Crystallography Core EM Tomography Core Eukaryotic Protein Expression Core Fluorescence Spectroscopy Core Protein Interactions Core Protein NMR Spectroscopy Core RNA Structure and Dynamics Core Tissue EM Core Virology Core Virus Imaging Core X-ray Crystallography Core |
EM Crystallography Core
Director: Mark Yeager, PhD Manager: Barbie Ganser-Pornillos, PhD The EM Crystallography Core focuses on: 1) Determining low to moderate-resolution structures of proteins and complexes that are not readily amenable to high-resolution NMR or X-ray crystallography studies, and 2) Acting as a salvage pathway for 3D crystal hits that have proven difficult to solve by X-ray crystallography. To perform these functions, Dr. Yeager has established the Molecular Electron Microscopy Core Laboratory, which includes multiple state-of-the-art microscopes for sample screening and data collection. Instrumentation and Capabilities Electron Microscopy The Molecular Electron Microscopy Core Laboratory includes an FEI Spirit 120 kV electron cryomicroscope, an FEI F20, and a Titan Krios. The Spirit is located within easy access on the second floor of the Snyder Building to screen samples for purity, homogeneity, and aggregation. The Spirit is equipped with a Gatan 2k x 2k CCD camera. The FEI F20 and Titan Krios will be installed and operational in early 2012 in a newly completed, specially designed EM suite within the Life Sciences Annex (LiSA). The LiSA EM core laboratory is located underground just adjacent to the Snyder Building, and is designed to minimize vibration, stray magnetic fields, and fluctuations in temperature and humidity. Both microscopes will be equipped with Gatan 4k x 4k CCD cameras. The Titan has an internal automatic cryogrid loading system, and the Spirit and F20 are used with Gatan 626 and 3500 cryostages. We also have a Nikon Super CoolScan 9000 ED scanner for digitizing electron micrographs. Until our F20 and Titan electron microscopes are installed, Dr. Edward Egelman has generously offered access to his group’s EM facility, located on the 6th floor of Jordan Hall, about 1.5 miles from Snyder. Dr. Egelman has Tecnai 120 kV and F20 electron cryomicroscopes. For the preparation and cryopreservation of EM grids, the laboratory houses a glow discharge unit (Electron Microscopy Sciences) and a Vitrobot (FEI). Protein Production The core is capable of large-scale culturing of bacteria, insect cells, and mammalian cells. We have access to three Innova 44R shakers (New Brunswick) dedicated to insect cell growth, a Vertiga IM shaker (Thomson Instruments) for small-scale insect cell expression experiments, and several AKTA chromotagraphy systems for analytical chromatography and preparative protein purification. X-ray Crystallography The UVa Dept. of Molecular Physiology and Biological Physics is fully equipped with an in-house X-ray crystallography facility. High-throughput crystallization is possible with a Mosquito liquid handling robotic system located in the Snyder Building, and we use a Rigaku MicroMax007 generator with a rotating Cu anode, an Osmic mirror optics system and a Saturn 92 CCD detector (Rigaku) for in-house screening. A helium compressor (X-Stream 2000) for generating gaseous nitrogen allows a constant supply of cryogen for low-temperature diffraction experiments. In addition, UVa has also purchased shares at the SER-CAT beamlines at Argonne National Laboratory to facilitate the collection of X-ray diffraction data. Structure Determinations The EM Crystallography Core screens proteins produced in-house and by other CHEETAH Cores for both in vitro assembly and/or 2D crystal formation. Low-resolution molecular envelopes resulting from these analyses are then used to build, test, and refine structural models and to design proteins more amenable to high-resolution studies. Examples of detailed methods that we have used in our analyses are provided in the following four publications from the current funding cycle: Ganser-Pornillos et al., Cell (2007) 131:70-79; Pornillos et al., Cell (2009) 137, 1282-1292; Ganser-Pornillos et al., Proc. Natl. Acad. Sci. (2011) 108:534-539; Pornillos et al., Nature (2011) 469:424-427. In brief, we screen purified proteins for assembly and/or 2D crystal formation using either monolayer crystallography or sparse screening of precipitants and buffers (similar to screening for 3D crystallography). Images of promising crystals or assemblies are acquired and analyzed by the MRC software package and/or 2dx. SITUS, Pymol, and Chimera are then used to build and visualize structural models for further testing.
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