Figure 1. Principle of the use of deuterated and selectively protonated protein in STD experiments (SOS‐NMR). These samples lead to positive STD at short saturation times only with ligand protons that are close to protonated residues. The only Ile near the binding site, Ile84, leads to saturation transfer to both rings of the ligand. In this example, the SOS data was sufficient to resolve problems in the protein–ligand complex structure determination due to the symmetry properties of the ligand.

Figure 2. General concept of the definition of restraints based on ambiguous NMR data in structural calculations using a physical force field. The following situation is presumed: an NMR experiment provides evidence that at least one of a group of protons in the protein (represented as orange dots) is adjacent to at least one of a group of protons in the ligand (represented as blue dots). An efficient distance d ^eff is then calculated with a weighting to the power of −6. This efficient distance is then included in Equation 1ab. As an effect, the additional potential stays quite flat as long as all atoms of the two groups are distinct to each other. However, the potential decreases steeply when at least two atoms come close to each other.

Figure 4. Illustration of the geometric parameters for the calculation of the electric field contribution (a), the bond anisotropy (b), and the ring current effect (c and d). Panels (c) and (d) illustrate the parameters using Equations 7 and 8, respectively.

Figure 3. Ring current‐induced CSPs based on Equation 8. (a) Orientation of the aromatic ring in (b) and (c). (b) Values for G(r) at potential protein proton positions 2 Å away from the benzene ring. The color of the surface encodes the value of G(r) at each position as defined in the color bar. (c) Same as (b), but 3 Å away from the ring.

Figure 5. (a) Schematic representation of the steps of an NMR screening project. The corresponding experimental NMR conditions are listed in Table 2. (b) Schematic representation of the NMR analysis of a library of compounds with NMR (orange frame in (a)). The procedure can be divided into three steps. The identification of binders by WaterLOGSY or STD experiments is usually followed by HSQC experiments that result in CSPs that readily allow the identification of the binding site when the protein is assigned. The last step is the identification of the binding mode, which is the most challenging, but often can be solved even with the CSPs alone. If the CSP data is not sufficient for the complex structure, additional data has to be collected and transferred into conformational restraints, which then lead to the complex structure. The variety of useful NMR parameters is summarized in Table 1.