Ultrahigh resolution protein structures using NMR chemical shift tensors

NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for ¹³Cαand ¹⁵N (peptide backbone) groups in a protein, the β1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific ¹³Cα and ¹⁵N CSTs were measured using synchronously evolved recoupling experiments in which ¹³C and ¹⁵N tensors were projected onto the ¹H- ¹³C and ¹H- ¹⁵N vectors, respectively, and onto the ¹⁵N- ¹³C vector in the case of ¹³Cα. The orientations of the ¹³Cα CSTs to the ¹H- ¹³C and ¹³C- ¹⁵N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8° . In addition, the measured ¹⁵N tensors exhibited larger reduced anisotropies in α-helical versus β-sheet regions, with very limited variation (18 ± 4° ) in the orientation of the z-axis of the ¹⁵N CSTwith respect to the ¹H- ¹⁵N vector. Incorporation of the ¹³Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance ¹³C- ¹⁵N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å(PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ ¹χ ² distributions, providing solid-state NMR with a powerful tool for de novo structure determination.