As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 μg in 8-35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of biomolecular NMR|
|State||Published - Jan 2010|
Bibliographical noteFunding Information:
Acknowledgments We thank A. Eletski, K. Singarapu, Y. Tang and R. Mani for helpful discussions, and for datasets used in the production of this manuscript. This work was supported by the National Institutes of General Medical Science Protein Structure Initiative program, grant U54 GM074958.
- Construct optimization
- NMR screening
- Structural genomics