Determining the three-dimensional structure of a small molecule-protein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D (1)H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.

Solid‐state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid‐state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1H‐detected assignment approaches, which correlate a given amide pair either to the two adjacent CO–CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross‐polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide‐protonated proteins from approximately 20 to 60 kDa monomer, at magic‐angle spinning (MAS) frequencies from approximately 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.