1 / 9

High-Throughput NMR Methods for Protein Structure Analysis

This document provides comprehensive reference and educational information on NMR data acquisition, specifically tailored for Varian and Bruker instruments. It covers expert techniques in NMR expected from users, standard and additional experiments for high-throughput NMR structure analysis, optimized experiments for stereo-specific assignment of isopropyl groups, histidine protonation state determination, and 3D NOESY experiments. Acknowledgements to key contributors included.

wesley-rasmussen
Download Presentation

High-Throughput NMR Methods for Protein Structure Analysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. NESG Wiki: NMR Data Acquisition Alexander Eletsky01/10/2010

  2. Introduction • Documenting NESG protocols: reference and educational information • Specific details for Varian and Bruker instruments • Some expertise in NMR expected from users HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  3. Spectromer Operation HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  4. NMR Experiments for Structure Determination • Standard experiment sets for high-throughput NMR • Additional experiments and novel methods HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  5. “Standard” NMR Experiments HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  6. Stereospecific Assignment of Leu/Val Isopropyl Groups • Optimized CT [13C,1H]-HSQC (28ms, 42ms and 56ms) for fractionally 13C-labeled samples D. Neri, T. Szyperski, G. Otting, H. Senn, K. Wuethrich, Biochemistry1989; 28(19):7510-7516 HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  7. HistidineProtonation State • Long-range [15N,1H]-HSQC or HMQC • Also yields He1/Ne1 spin assignments J. G. Pelton, D. A. Torchia, N. D. Meadow, and S. Roseman, Protein Sci. 1993; 2(4): 543–558. HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  8. 3D 13C/15N-resolved NOESY • Optimized for simultaneous 13C(aliphatic), 13C(aromatic) and 15N frequency labeling • Uses adiabatic 13C decoupling sideband suppression (SEAD – E. Kupce) • Spectra acquired at 900 MHz • Three-fold time saving compared to separate experiments S. M. Pascal, D. R. Muhandiram, T. Yamazaki, J. D. Forman-Kay, and L. E. Kay, JMR Ser. B. 1994; 103:197–201.Y. Shen, H. S. Atreya, G. Liu, and T. Szyperski, JACS 2005; 127:9085-9099. HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

  9. Acknowledgements • James M. Aramini • AleksandrasGutmanas • Gaohua Liu • Jeffrey L. Mills • Theresa A. Ramelot • Paolo Rossi • Kiran K. Singarapu • Yibing Wu • DuanxiangXu • Qi Zhang HTP Methods for Protein NMR Structure Analysis Used by the NIGMS Protein Structure initiative, January 10, 2010

More Related