Atomic resolution structure of Escherichia coli dUTPase determined ab initio

Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffra ct to atomic resolution. Data to 1.05 Angstrom resolution were collected fr om a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site pe r subunit of the trimeric enzyme) using the program ARP/ wARP. After refine ment with anisotropic temperature factors a highly accurate model of the ba cterial dUTPase was obtained. Data to 1.45 Angstrom from a native crystal w ere also collected and the 100 K structures were compared. Inspection of th e refined models reveals that a large part of the dUTPase remains rather mo bile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site c avity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-bind ing site. Comparison between the native enzyme and the mercury complex show s that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially usef ul tool for further studies of inhibitor- substrate-analogue complexes of t his protein at very high resolution.