Solution NMR is a powerful tool for structural characterization of proteins and protein complexes not exceeding 200-300 amino acid residues, but difficulties in obtaining large quantities of properly refolded, isotopically enriched protein make this approach challenging for the m4 protein. However, we have been able to obtain virtually complete backbone resonance assignments as well as limited amide-amide NOE data for this system. Recent advances in modeling techniques using the program Rosetta in principle make it possible to model the structure of the protein on the basis of this chemical shift and limited NOE information. The physically realistic energy function implicit to Rosetta can discriminate native from non-native protein conformations and the chemical shift and NOE information strongly reduce the permissible region of conformational space. Early stage models for m4 have been derived from this approach, and these are currently subjected to newly developed validation methods in order to evaluate how unambiguous the obtained structure is.

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