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 |
Protein NMR Spectroscopy Core
Director: Wes Sundquist, PhD Manager: Jack Skalicky, PhD The Protein NMR Spectroscopy Core determines high-resolution structures of proteins and complexes, and analyzes their dynamics. We have excellent access to multiple state of the art, high-field NMR spectrometers, including one 600 MHz, two 800 MHz and one 900 MHz instruments (described below). Pulse sequences and software paths for structure determinations have been implemented and tested extensively. Instrumentation and Capabilities Utah - Varian Inova 600 NMR Spectrometer Our Laboratory for Biomolecular NMR Spectroscopy houses a Varian Inova 600 MHz NMR spectrometer operated by a Linux Dell Precision T3500 computer. The spectrometer has three complete RF channels, a fourth dedicated 2H channel, and is equipped with a triple resonance (1H,13C,15N) cryogenic probe with Z-axis pulsed field gradient (PFG) capability. Following acquisition, primary data is transferred to our Structural Biology Computing Center (SBCC) for processing and analysis. The SBCC contains three PowerServe Duo A21200 clients (8, 16, & 24 cpu) and three LINUX Dell Precision 670 clients (2 cpu). A Dell PowerEdge 640 (3 Tbyte) server provides centralized data storage. Core members are the major users of this instrument, which is operated on a cost reimbursement basis with a University subsidy, which covered part of the salaries for Dennis Edwards (hardware technician) and Jack Skalicky (Director), cryogens, cryogenic probe yearly maintenance, and non-consumables. Utah - Varian DirectDrive 800 MHz NMR Spectrometer We are also members of the new David M. Grant NMR Center (www.nmrc.utah.edu) (Peter Flynn, Director), which provides us with full access to a Varian DirectDrive 800 MHz NMR spectrometer. The instrument has four RF channels, Z-axis PFG, and a triple resonance probe. We pay $160/day for use of this spectrometer. Colorado - Varian 800 and 900 MHz NMR Spectrometers In addition to our on-site spectrometers, we have: 1) 30% of the instrument time on a Varian DirectDrive 800 housed in Boulder, Colorado (http://cunmr800.colorado.edu/), purchased as part of a shared instrumentation grant (PI, Arthur Pardi, UC Boulder), and 2) ~10% of the time on a Varian DirectDrive 900 housed in Denver, Colorado (http://cunmr800.colorado.edu/900home.html), purchased by our Rocky Mountain NMR consortium. Both instruments are "fully loaded" and include four complete RF channels, XYZ-axis PFG, and salt-tolerant, carbon-enhanced triple resonance cryogenic probes. Data are typically collected remotely, and we primarily use these instruments to record NOESY experiments and TROSY-based experiments. We pay an annual “buy-in” fee for the 800 and 900 of $4600/yr, plus a usage fee of $138/day for either spectrometer. Protein Structure Determinations and Dynamics Analyses Detailed methods and full references for protein complex structure determinations are provided in two Core publications from the previous funding cycle (Stuchell-Brereton et al., Nature (2007) 449:740-4; Kieffer et al. Dev. Cell (2008) 15:62-73) and one from the current funding cycle (Skalicky et al., JBC (2013), in press). Briefly, isotopically-labeled proteins (1H,13C,15N, and 2H as necessary) are typically made in E. coli. Core triple resonance data sets are collected and used as input for the automated assignment software program, AutoAssign. Distance restraints for structure calculations are obtained from a single 3D [1H,13C/15N,1H] NOESY which is processed to yield separate 13Cali+aro and 15N edited NOESY spectra. Backbone torsion angle restraints are obtained from chemical shifts using the software TALOS+, and dipolar coupling restraints are obtained from proteins in aligned and isotropic media and analyzed within SPARKY. We have implemented the following software “path” for offline data processing, spectral assignments, structure calculations and analysis: FELIX ® SPARKY ® AUTOASSIGN ® CYANA® XPLOR-NIH ® PYMOL® ADIT-NMR Structure Validation (All software is LINUX compatible or web-based). Detailed methods for protein dynamics studies are provided in recent publications from Skalicky and colleagues: (Davulcu et al., Structure (2009) 17: 1356-67; Niu et al., J. Mol. Biol. (2011) 405:479-96, and Davulcu et al., Biochemistry (2011) 50: 4011-18).
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