New methods of structure refinement for macromolecular structure determination by NMR

Abstract

Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13C shifts, 1H shifts, T1/T2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters.

Figure 1

Correlation between backbone precision of NMR structures and their agreement with x-ray structures. Where the backbone rms difference between the average NMR coordinates (NMR) and the corresponding x-ray structures is available, the values are represented as circles. When only the average backbone rms difference between an ensemble of NMR structures () and the corresponding x-ray structure is quoted in the literature, squares are used. The straight line represents a linear fit to the data with a slope of 0.70, an intercept of 0.45 Å, and a correlation coefficient of 0.9. The structures are as follows: p53(mon), p53(dim), and p53(tet) are the monomer, dimer, and tetramer, respectively, of the p53 oligomerization domain (51); IL-8, interleukin-8 monomer (52); Hir (new), highly refined structure of hirudin (53); IL-1, interleukin-1β (6, 7); BPTI, bovine pancreatic trypsin inhibitor (54); eglin c (55); PC, French bean plastocyanin (56); tendamistat (57); Hir(old), hirudin (58); Cyp-CsA, cyclophilin–cyclosporin A complex (59); Mb, carbonmonoxy myoglobin (helices plus heme; ref. 60); CPI, potato carboxypeptidase inhibitor (61); PCP-B, procarboxypeptidase B (62); and BSPI, barley serine proteinase inhibitor 2 (63). The values given exclude conformationally disordered regions as described in the papers cited. Note that the NMR structures of IL-8 and Hir(old) were obtained before the corresponding x-ray structures and that the NMR structure of tendamistat was obtained independently of and at the same time as the x-ray structure. Reproduced from ref. .

Figure 2

View of the active site and neighboring regions of reduced human thioredoxin showing a superposition of 40 simulated annealing structure before (blue) and after (red) ¹H chemical shift refinement. Reproduced from ref. .

Figure 3

View showing bestfit superpositions of the restrained regularized mean coordinates obtained with and without dipolar coupling restraints. The protein is shown as a ribbon diagram drawn through the C^α positions. The loop between strands β3 and β4 (residues 21–24) is shown in magenta for the structure obtained with dipolar coupling restraints and in grey for the structure obtained without dipolar coupling restraints. Adapted from ref. .