Direct nuclear overhauser effect refinement of crambin from two- dimensional NMR data using a slow-cooling annealing protocol

The solution structure of crambin has been refined using a direct nuclear Overhauser effect (NOE) simulation approach (DINOSAUR) following a slow- cooling simulated annealing protocol starting from eight previously obtained nmr and the x-ray structures of crambin. Theoretical NOE intensities calculated with inclusion of local motions were directly compared to the experimental nmr data and forces were derived using a simple first-order approximation for the calculation of the NOE gradient. A dynamic assignment procedure was applied for the peaks involving unassigned diastereotopic proton pairs or equivalent aromatic protons. With this approach, R factors could be minimized in a reasonable simulation time to low values (around 0.26) while deviations from ideal bond lengths and angles are still acceptable. The improvement in R factors is accompanied by an improvement of the precision of the structures, the rms deviations (rmsd; from the average) calculated on the ensemble of nine structures decreasing from 0.65 to 0.55 Å for backbone atoms and from 1.0 to 0.85 Å for all heavy atoms. The solution structure is significantly different from the x-ray structure with rmsd for all atoms of 1.35 Å compared to 0.85 Å between solution structures. The largest differences are found for residues Thr-21 and Pro-22 in the loop region between the two α-helices and for the side chain of Tyr-29.