Protein production and characterization
The Institute is well-equipped with a wide array of instrumentation for cloning, and heterologous protein overexpression facilities for bacteria, yeast, insect, and mammalian cell lines, including incubators, centrifuges, ultracentrifuges, freezers, and balances. For sample purification and characterization, we have HPLCs with UV-Vis, fluorescence, and light-scattering detectors, several FPLC systems, a differential scanning fluorimeter, plate readers with dynamic light-scattering, UV-Vis absorption, fluorescence, and bioluminescence detection, a surface plasmon resonance instrument, an isothermal titration calorimetry instrument, UV-Vis spectrophotometers, and multiple gel imager stations that enable visible, fluorescence, and chemiluminescence imaging. For studies of membrane proteins, we are equipped to reconstitute them in vesicles and bead-supported unilamellar membranes for functional assays, perform electrical recordings from proteins in planar lipid bilayers and bead-supported unilamellar membranes, and patch-clamp recordings from ion channels in cultured and primary cells. Biophysical characterization capabilities include an XtalConcepts SpectroLight 600 DLS with imaging and plate reading capabilities; and Stratogene Mx3005P real-time PCR for DSF studies. We also have a synthetic chemistry laboratory, with a Schlenk manifold, high vacuum pumps, a rotary evaporator, a lyophilizer, and a peptide synthesis station. A dedicated radiation facility houses a liquid scintillation counter. Instrumentation is shared between individual investigators so that investment in any single instrument benefits the scientific endeavor at the institute as a whole.
The Institute has a dedicated Cryo-electron Microscopy (cryo-EM) Center consisting of dedicated microscope facilities with precise environmental control that minimizes temperature drift or fluctuation, electromagnetic interference, mechanical vibrations, and acoustic noises. An energy storage system provides low noise electrical power and several hours of backup for the cryo-EM facility and most of the Institute. The facility has a dedicated control room of the microscopic systems, a sample preparation laboratory with a low humidity room and a visitors’ lounge for collaborators. Remote data viewing is available and the facility is rounded out by dedicated computational and storage servers. The facility has a 200 kV Glacios Cryo transmission electron microscope with Falcon 4 and Ceta-D detectors and is equipped to perform both single-particle and microcrystal electron diffraction studies. The center is configured with facilities able to support up to three microscopes including a 300 kV system.
The Cryo-EM Center works with academic and commercial users providing cryo-EM services including sample preparation, characterization, data collection, and analysis for high-resolution structural studies.
High-Throughput Crystallization Screening Center
The Institute is home to the High-Throughput Crystallization Screening Center. The Crystallization Center works with academic, government, non-profit, and commercial users providing crystallization screening services.
The Center has run crystallization screening experiments on almost 20,000 macromolecules from over 1,000 laboratories worldwide. In the standard mode, crystallization screening takes place in 1,536 different chemical conditions using the microbatch-under-oil method. Instrumentation to accomplish this includes an Art Robbins Gryphon with LCP, Thermo Scientific Matrix PlateMates, Integra ViaFlo 384, and Hydra DT liquid handling systems, as well as a Robbins Scientific Tango liquid handling system. The Center is also equipped to set up vapor diffusion crystallization screening experiments and for the optimization of crystallization conditions.
Crystallization plate storage and incubation include three Precision Scientific Incubators and four temperature-controlled rooms devoted exclusively to crystallization across a range of temperatures. An ultralow temperature -86°C Revco Upright Freezer is available to store recovered sample aliquots following the setup of crystallization screening experiments.
The ability to detect crystals buried beneath precipitate and to detect crystals that are too small to see using standard brightfield microscopy is a major asset of the imaging capabilities of the Crystallization Center. To identify chiral crystals, including submicron-sized crystals, and crystals buried in the precipitate, a state-of-the-art Formulatrix Rock Imager 1000 with SONICC has been modified in collaboration with Formulatrix for our high-throughput application. The Crystallization Center is the only centralized resource in the United States that makes the Rock Imager with SONICC and its capabilities available to a wide user community of structural biology researchers. The brightfield imaging, along with the ability to detect second-harmonic generation (SHG) from chiral crystals and UV-Two Photon Excited Fluorescence (UV-TPEF) from concentrated protein is used by the Crystallization Center to identify crystallization conditions under which crystals were too small to see by microscopy and thus would have been missed, or classified as precipitates without SONICC. The Rock Imager 1000, as modified, has the capacity to image 5 x 1,536 well plates in a 24-hour period with visible, SHG, and UV-TPEF. A second Formulatrix Rock Imager 54 (without SONICC) is located in a temperature-controlled room adjacent to the Crystallization Center robotics laboratory and allows the exploration of temperature as a variable in crystallization.
A robust laboratory information system supports the Crystallization Center which includes the backup of imaging data. The Center has been a major contributor to the MAchine Recognition of Crystallization Outcomes (MARCO) project and is continually at the forefront of the development of computational resources for the analysis of images and crystallization conditions.
The High-Throughput Crystallization Screening Center is a core facility for determining initial crystallization conditions, providing rapid screening of 1,536 chemical crystallization conditions. In 20 years of operation, the Crystallization Center has set up over 25 million experiments on more than 17,000 samples. Instrumentation includes an Art Robbins Gryphon with LCP, Thermo Scientific Matrix PlateMates, Integra ViaFlo 384, and Hydra DT liquid handling systems, as well as a Robbins Scientific Tango liquid handling system, for the generation of crystallization plates. We have a Formulatrix Rock Imager 1000 with SONICC (SHG and UV-TPEF) at 23°C, a Rock Imager 54 at 14°C, and a robust imaging schedule for automated imaging. See www.getacrystal.com for more details.
X-Ray diffraction facilities include a high-flux rotating anode generator with focusing optics and a CCD detector. A cryogenic cooling stream is available. The laboratory facilities are supplemented by the availability of time at National Laboratories including Brookhaven National Laboratories, Argonne National Laboratory, Stanford Linear Accelerator Center, and the Cornell High Energy Synchrotron Source. Institute scientists use facilities at all these laboratories and the laboratory runs the IMCA beamline on the Advanced Photon Source at Argonne National Laboratory. Beamtime is available on the IMCA beamline through a subscription process. Scientists also collect SAXS data in Stanford and neutron data at the Oak Ridge National Laboratory.