3.6.7. Nuclear Magnetic Resonance Molecular Replacement (NMR2)
The most recent approach called Nuclear Magnetic Resonance Molecular Replacement (NMR2) utilizes spatial data obtained through solution-state NMR in order to locate the binding pocket of a complex structure. For that, it uses a receptor model, e.g., a X-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment. To conduct an experiment using such an approach requires a few steps. First, either the protein or ligand used in the complex must be uniformly 13C and 15N labeled. Then, an experiment to assign the ligand is needed such as 2D 13C,1H-HMQC or 13C,1H-HMBC. The next step is the evaluation of ligand intra- and ligand–protein intermolecular distances through NOE cross peaks obtained from F1-15N,13C-filtered 1H,1H-NOESY. Lastly, choosing a proper input structure is required which can be either X-ray or NMR structures in apo form, with another bound ligand, or a homolog to the protein of interest. Then the NMR2 program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [403,404]. This method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [403,404,405,406].