National Magnetic Resonance Facility at Madison

National Magnetic Resonance Facility at Madison

Research Emphasis

The National Magnetic Resonance Facility at Madison (NMRFAM) develops multinuclear, multidimensional nuclear magnetic resonance (NMR) approaches to solution-state studies of biological macromolecules, such as proteins, nucleic acids, carbohydrates, and lipids. Related collaborative projects converge on structure-function investigations of proteins and nucleic acids, structural genomics, and metabolomics. The resource has considerable experience in labeling proteins and RNA with stable isotopes and in studying chemical and dynamic properties of biomolecules, such as binding equilibria, conformational equilibria, internal mobility, and protonation steps. Data collection for oxygen-sensitive samples and proteins containing paramagnetic centers is a specialty. NMRFAM develops methods for efficient NMR data collection, processing, structure determination, validation, and data deposition. Computational resources include not only hardware and software but also expertise in ab initio calculations of chemical shifts and J-couplings from structural modeling.

Current Research

Fast data collection and automated data analysis. Technology for larger proteins and complexes. Metal-containing (paramagnetic) proteins. Dynamics of macromolecules. Structure-function investigation of RNA molecules and their complexes with metal ions and proteins.

Resource Capabilities

Methods

NMRFAM offers start-to-finish support for biomedical NMR investigations conducted at the facility or remotely over the Internet. As needed, NMRFAM will lend support to one or more of the following steps: (1) strategy evaluation, (2) sample preparation, (3) feasibility studies, (4) data collection, and (5) data analysis and structure determination. Our aim is to facilitate the efficient pursuit of new knowledge by providing researchers with resources matched to their particular needs.

Instruments

Nine NMR spectrometers, all with variable temperature, ²H lock, pulsed-field gradient, and shaped pulse capability. 900-MHz (21.1 T/54 mm) Unity Inova NMR spectrometer with ¹H-detection triple-resonance ColdProbe, 800 (18.8 T/63 mm) Unity Inova NMR spectrometer with ¹H-detection triple-resonance ColdProbe, Bruker DMX 750-MHz (17.6 T/52 mm) NMR spectrometer with ¹H-detection triple-resonance CryoProbe (with cold ¹³C preamp), Bruker DMX 600 MHz spectrometer (14.1 T/52 mm) with ¹H-detection triple-resonance CryoProbe, 600 (14.1 T/51 mm) Unity Inova NMR spectrometer with ¹H-detection triple-resonance ColdProbe and with an automated sample changer, 600 (14.1 T/51 mm) Varian NMR system (VNS) spectrometer with ¹H-detection triple-resonance ColdProbe, Bruker DMX 500-MHz spectrometer (11.7 T/52 mm) with ¹H-detection triple-resonance CryoProbe, Bruker DMX 500-MHz spectrometer (11.7 T/52 mm) with an assortment of standard probes, Bruker DMX 400 MHz (9.4 T/89 mm) with a ¹³C, ¹⁵N, ³¹P, ¹H Quattro Nucleus Probe and an assortment of other standard probes including microimaging and surface coil.

Software

Software developed in-house includes PISTACHIO (probabilistic automated assignments), PECAN (probabilistic secondary structure prediction from NMR data), and LACS (method for checking protein NMR datasets for anomalous values or potential referencing problems). These software packages are available to all through a Webserver accessed from the NMRFAM home page. Software for the implementation of rapid data collection methodology (HIFI-NMR) is available on NMRFAM spectrometers. Pulse programs and other software developed as NMRFAM are freely available for research purposes, either by distribution or from efficient local servers.

Special Features

NMRFAM provides young investigators and experienced spectroscopists access to state-of-the-art instrumentation with support for multiple modes of data collection. Protocols, pulse sequences, and software tools developed through NMRFAM's research activities are made available to the general scientific community. The aim is to develop and disseminate advanced approaches to experiment design and data analysis that cover all steps in a biomolecular NMR investigation, from cloning through data deposition. Much of the training at NMRFAM is carried out one-on-one and hands-on with a facility staff member in the course of data collection. NMRFAM conducts workshops and group training sessions; these are announced on the Web site.

Available Resources

A small laboratory is available to outside users of the facility. Its equipment includes a pH meter, NMR tube centrifuge, lyophilizer, refrigerator and freezer, fume hood, and balance. Desks and workstations are available for short-term visitors, and an office is available for longer-term visitors. In addition to the NMR instrumentation described above, NMRFAM makes its computational resources available to users. Software packages from both commercial and academic sources are available for the analysis of NMR data, computation of structures, and quantum mechanical calculations. Available programs include nmrPipe, Felix, Sparky, X-Plor-NIH, Cyana, Amber, Dock 5, Gaussian 2003, Amsterdam Density Functional, and Octave 2.1.50.

Training Opportunities and Workshops

Much of the training at NMRFAM is carried out one-on-one and hands-on with a facility staff member in the course of data collection. NMRFAM holds workshop/training sessions that concentrate on new technology and new methodology. These are aimed at the level of graduate students, postdocs, and scientists from other disciplines interested in biomolecular NMR. The sessions involve invited outside speakers and NMRFAM staff members. They are announced on the NMRFAM Web site, sent to subscribers of the NMRFAM newsletter, and advertised at national and international conferences. 2006 Workshops: "NMR Data Collection and Analysis for Biological Structure Determination" (06/06-09) and, joint with the Center for Eukaryotic Structural Genomics, "Wheat Germ Cell-Free Protein Production" (07/30-08/04).

Publications

1. Eghbalnia, H. R., Bahrami, A., Wang, L., Assadi, A., and Markley, J. L. Probabilistic identification of spin systems and their assignments including coil-helix inference as output (PISTACHIO). Journal of Biomolecular NMR 32:219–233, 2005.

2. Eghbalnia, H. R., Bahrami, A., Tonelli, M., Hallenga, K., and Markley, J. L., High resolution iterative frequency identification for NMR (HIFI-NMR) as a general strategy for multidimensional data collection. Journal of the American Chemical Society 127:12528–12536, 2005.

3. Blad, H., Reiter, N. J., Abildgaard, F., Markley, J. L., and Butcher, S. E., Dynamics in the U6 RNA intramolecular stem loop: II. NMR analysis of metal binding and conformational exchange on the microsecond time scale. Journal of Molecular Biology 353:540–555, 2005.

4. Lin I.-J., Gebel, E. B., Machonkin, T. E., Westler, W. M., and Markley, J. L., Changes in hydrogen bond strengths explain variations in the reduction potentials in a series of rubredoxin mutants. Proceedings of the National Academy of Sciences USA 102:14581–14586, 2005.