Bacterial Protein Expression Core
Cell Biology and Imaging 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
Virus Imaging Core
X-ray Crystallography Core
RNA Structure and Dynamics Core
Director: Elisabetta Viani Puglisi, PhD
Co-Director: Jody Puglisi, PhD
The RNA Structure and Dynamics Core provides expertise and capabilities in RNA synthesis and purification for structural and biophysical studies. Resources include instrumentation for NMR spectroscopy (900, 800, 600 and 500 MHz spectrometers), X-ray crystallography, and single-molecule fluorescence and force measurements.
Instrumentation and Capabilities
Core workers have full access NMR spectrometers housed in the Stanford Magnetic Resonance Laboratory (SMRL) (J. Puglisi, Director; Dr. Corey Liu, Chief Staff Scientist). SMRL houses a Bruker 500 MHz NMR spectrometer equipped with a triple-resonance cryoprobe, a Varian Inova 600 MHz spectrometer equipped with a variety of room temperature probes, and a Varian 800 MHz spectrometer with a recently upgraded DirectDrive console, equipped with a triple resonance cryoprobe. NMR time is charged at an hourly rate, generating income for the SMRL service center. Additional funds are supplied by the Stanford University School of Medicine, which subsidizes the salary and gas cryogen costs for the three NMR spectrometers. The Core also has access (1 day/week) at the UC Berkeley Bruker 900 MHz facility.
In addition to facilities within the X-ray Crystallography Core, the RNA Structure and Dynamics Core has on-site access to a Phoenix Crystallization robot for crystal screening, and access to beamlines at Stanford Synchotron Radiation Laboratory (SSRL), which provides beamline 11.1 for Stanford faculty.
Biochemistry, Sample Preparation, and Characterization
The Core is equipped with standard biochemical equipment including a Pharmacia FPLC system for both protein and RNA purification, a Varian HPLC and preparative gel electrophoresis. We currently purify both RNA and proteins on the same FPLC system, and we are therefore requesting funds toward the purchase of a new AKTAPurifier10 FPLC system that will be dedicated to RNA purification. The Core also has a VP Isothermal Titration Calorimeter for determination of RNA-protein thermodynamic parameters.
The Core contains a three color, single-molecule total internal reflectance (TIRF) system with illumination at 488, 532, and 642 nm, and Quadview detection of up to 4 colors. The system is equipped with an EM-CCD camera capable of 20ms time resolution, which allows rapid mixing of two reagents. Data are processed with custom-written software that is available to the community.
The core also houses an optical trapping system for single-molecule force measurements. The system can perform simultaneous force and two-color fluorescence measurements.
Finally, the Core has access to single-molecule instrumentation for acquisition of data within Zero mode waveguides (ZMW) through Pacific Biosciences. The investigators have 1 day/week access on instrumentation for 3-color excitation, 4/5-color detection of single-molecule fluorescence binding events.
Biophysical Methods and Analyses
Detailed methods for NMR structural studies are presented in our recent papers by Viani Puglisi (J. Mol Biol. (2011) 410(5):863), and reviews in past years (Lukavsky & Puglisi, Methods Enzymol; (2006) 394:399-41). Briefly, we use standard heteronuclear and homonuclear NMR experiments for assignments, and NOE, dihedral and residual dipolar coupling measurements for structure determination. For single-molecule analysis, we have developed a number of methods for analyzing both conformational (single-molecule FRET) and compositional (ZMW) dynamics (Uemura et al., Nature (2010) 464:1012-7; Aitken et al., Nat Struct Mol Biol. (2010) 17:793-800).