J. Mol. Biol. (1988) 264, 675-724 Determination of the Complete Three-dimensional Structure of the a-Amylase Inhibitor Tendamistat in Aqueous Solution by Nuclear Magnetic Resonance and Distance Geometry Allen D. Kline, Werner Braun and Kurt Wiithrich Institut $iir Molekularbiologie und Biophysik Eidgen&sische Technische Hochschule-Hiinggerberg CH-8093 Ziirich, Switzerland (Received 18 March 1988) The complete three-dimensional structure of the a-amylase inhibitor Tendamistat in aqueous solution was determined by ‘H nuclear magnetic resonance and distance geometry calculations using the program DISMAN. Compared to an earlier, preliminary determination of the polypeptide backbone conformation, stereo-specific assignments were obtained for 41 of the 89 prochiral groups in the protein, and a much more extensive set of experimental constraints was collected, including 842 distance constraints from nuclear Overhauser effects and over 100 supplementary constraints from spin-spin coupling constants and the identification of intramolecular hydrogen bonds. The complete protein molecule, including the amino acid side-chains is characterized by a group of nine structures corresponding to the results of the nine DISMAN calculations with minimal residual error functions. The average of the pairwise minimal root-mean-square distances among these nine structures is 0.85 A for the polypeptide backbone, and 1.52 A for all the heavy atoms. The procedures used for the structure determination are described and a detailed analysis is presented of correlations between the experimental input data and the precision of the structure determination. - 1. Introduction In the development of nuclear magnetic resonance (n.m.r.t) in solution as a second method for protein structure determination besides X-ray diffraction in single crystals (Withrich, 1986), the project with the a-amylase inhibitor Tendamistat has a special place. It was initiated in 1985 after the first structure determinations using n.m.r. and distance geometry (Blumenthal, 1970; Crippen, 1981) had been completed with the polypeptide hormone glucagon (Braun et al., 1981, 1983) and the globular protein BUSI IIA (Williamson et al., 1985), when doubts were raised about the feasibility of t Abbreviations used: n.m.r., nuclear magnetic resonance; BUS1 IIA, protcinase inhibitor IIA from bull seminal plasma; 2&F-COSY, S-quantum filtered Z-dimensional spectroscopy; NOESY, Z-dimensional NOE spectroscopy; COSY, Z-dimensional correlated spectroscopy; p.p.m., parts per million; NOE, nuclear Overhauser enhancement; RMSD, root-mean-square deviation; Tendamistat, a-amylase polypeptide inhibitor from Streptomyces tendae. such structure determinations without access to related crystal structures. Upon the suggestion of Dr R. Huber, structure determinations of Tendamistat were started simultaneously and completed independently in his laboratory using X-ray methods with single crystals, and in our laboratory using n.m.r. in solution, The initial comparisons for the polypeptide backbone (Kline & Wiithrich, 1985; Kline et at., 1986; Pflugrath et al., 1986) showed that the same global molecular architecture of Tendamistat was observed in single crystals by X-ray diffraction and in aqueous solution by n.m.r. This observation has undoubtedly had impact on the general acceptance of n.m.r. as a method for protein structure determination in solution (Kabsch & Rijsch, 1986), even though it had previously, a priori, been clear that protein structure determinations by n.m.r. in solution do in no way have to rely on prior knowledge of the corresponding crystal structure, or could receive much help from the availability of such information (Wiithrich et al., 1982; Have1 & Wiithrich, 1984, 1985; Braun & Go, 1985; Williamson et al., 1985; Wiithrich, 1986). In the 0022-2S36/88/230675-50 $03.00/O 675 0 1988 Academic Press Limited