Featured Technology Archive

Leveraging the Power of Molecular Fragment Replacement to Solve Protein Structures by NMR

2JPZ The protein structures deposited in the PDB during the Protein Structure Initiative (PSI) have been determined by x-ray crystallography and NMR spectroscopy. While the majority of these structures were solved using x-ray crystallography, nearly 500 were solved using NMR. NMR spectroscopists determine protein structure by assigning chemical shifts to the backbone atoms and deriving distance and global structural information from nuclear Overhauser enhancements (NOEs). In some cases, additional information is needed to accurately solve the three-dimensional protein structure such as experimentally determined distance or orientation restraints.

To enhance this information, the experimental data can be used to search the PDB for compatible regions of structures and build a 3D structure model in a method called molecular fragment replacement (MFR). MFR was first employed in x-ray crystallography where molecular fragments were built to fit experimentally determined electron density (Jones and Thirup, 1986). The first application to NMR was demonstrated by in the laboratory of Ad Bax (Delaglio F, et al., 2000). Within the PSI, it has been applied to solving NMR structures of water-soluble proteins in conjunction with CS (chemical shift)-Rosetta by a collaborative effort of the PSI:Biology High-Throughput Center Northeast Structural Genomics consortium (NESG; Guy Montelione, PI) in 2008 (Shen, et al., 2008). This application was originally proposed as a way to automate structure determination, and therefore to decrease the time from target selection to structure determination. Building on their previous work, NESG incorporated orientation restraints - residual dipolar couplings (RDCs) - into the CS-Rosetta structure prediction method and used it to successfully solve structures with backbone-only NMR data (Raman, et al., 2010).

More recently, one of the PSI:Biology Membrane Centers, Membrane Protein Structures by Solution NMR (MPSbyNMR), under the direction of James Chou, PhD, at Harvard Medical School, combined NMR and MFR to solve the structure of the 300-residue transmembrane receptor murine mitochondrial uncoupling protein 2 (UCP2; Berardi MJ et al., 2011). 2LCK In this study, the UCP2 sample was reconstituted in micelles for solution NMR experiments. Typically, NOEs are assigned, but in this case, it was difficult to gather enough unambiguous NOEs due to backbone resonance overlap. Further orientation information was needed, so RDCs were measured. Using this experimental data, researchers employed the MFR method, searching the Protein Data Bank to construct a database containing over 300,000 seven-residue fragments and selecting the ones with the best fit. These fragments were then used to determine backbone structure using a four-step procedure to assign corresponding protein segments without overlapping, filling gaps, and extending the fragments. Fifteen structured segments were identified through this molecular fragment searching method and used in the subsequent structure calculations. These data, in addition to experimentally determined distance restraints, allowed the researchers to solve the solution structure of this protein and gain information about the region of substrate binding to this integral membrane protein.

The successful application of this technique to the study of this large transport protein will enable its use to discover the mechanisms of other membrane-embedded transporters and translocators. In the future, researchers hope to capitalize on the power of NMR and MFR to gain insight into the biological function of these molecules by probing the conformational changes coupled to substrate transport.

Berardi MJ, et al. “Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching.” Nature 476:109-114 (2011).

Delaglio F, Kontaxis G, Bax A. "Protein structure determination using molecular fragment replacement and NMR dipolar couplings." J. Biomol. NMR 38:289-302 (2000).

Jones TA and Thirup S. “Using known substructures in protein model building and crystallography.” EMBO J. 5:819-22 (1986).

Raman S, et al. “NMR structure determination for larger proteins using backbone-only data.” Science 327:1014-1018 (2010).

Shen Y, et al., “Consistent blind protein structure generation from NMR chemical shift data.” PNAS 105:4685-4690 (2008).


NMR solution structure of ALG13: The sugar donor subunit of a yeast N-acetylglucosamine transferase. Northeast Structural Genomics Consortium target YG1. 2JCZ DOI:10.2210/pdb2jzc/pdb


Structure of the mitochondrial uncoupling protein 2 determined by NMR molecular fragment replacement. 2LCK DOI:10.2210/pdb2lck/pdb

Relevant PSI SBKB Technical Highlights

July 2010: Nuclear magnetic resonance spectroscopy
A general overview of PSI efforts in the realm of protein structure determination using NMR. This article contains additional information about automated NMR structure determination and mentions CS-Rosetta and CS-RDC-Rosetta.

September 2011: A fragmented approach to membrane protein structures
A more technical description of the work done by MPSbyNMR combining MFR with NMR.

Looking for more membrane protein-related resources? Go to the PSI SBKB Membrane Proteins Hub.

share on Facebook Tweet This share on LinkedIn add Google Bookmark add to delicious Share on reddit share on StumbleUpon Digg this