Abstract
The structure of Chromatium high potential iron protein (HiPIP) has been refined by semiautomatic Fo-Fc (observed minus calculated structure amplitude Fourier methods to a convential R index, R=sum of the absolute value of Fo-Fc divided by the sum of Fo, of 24.7% for a model in which bond distances and angles are constrained to standard values. Bond length and angle constraints were applied only intermittenly during the computations. At a late stage of the refinement, atomic parameters for only the Fe4S4 cluster plus the 4 associated cystein S-gamma atoms were adjusted by least squares methods and kept fixed during the rest of the refinement. The refined model consists of 625 of the 632 nonhydrogen atoms in the protein plus 75 water molecules. Seven side chain atoms could not be located in the final electron density map. A computer program rather than visual inspection was used wherever possible in the refinement: for locating water molecules, for removing water molecules that too closely approach other atoms, for deleting atoms that lay in regions of low electron density, and for evaluating the progress of refinement. Fo-Fc Fourier refinement is sufficiently economical to be applied routinely in protein crystal structure determinations. The complete HiPIP refinement required approximately 12 hours of CDC 3600 computer time and cost less than $3000 starting from a "trial structure," based upon multipe isomorphoous replacement phases, which gave an R of 43%...
Highlights
F,F, Fourier methods to a conventional R index, R = c 1F,F, 1/c F, of 24.7% for a model in which bond distances and angles are constrained to standard values
Seven side chain atoms could not be located in the final electron density map
A computer program rather than visual inspection was used wherever possible in the refinement: for locating water molecules, for removing water molecules that too closely approached other atoms, for deleting atoms that lay in regions of low electron density, and for evaluating the progress of refinement
Summary
The structure of Chromatium high potential iron protein (HiPIP) has been refined by semiautomatic. (d) l:recise atomic parameters are required for computing the best possible phases from which, in turn, the clearest possible difference-electron-density maps may be calculated The power of this difference technique, as applied, for example, to the study of substrate-analog and inhibitor binding (9, IO) has made it an invaluable tool for understanding enzyme function. During the short history of protein crystallography, man) different refinement techniques have been applied in order to enhance the clarity of electron density maps and to obtain more accurate protein models: conventional F, Fourier refinement (11); model-building methods in which the protein model is adjusted so that its bond lengths and bond angles conform to standard values obtained from crystal structures of the cow stituent amino acid? The successful refinement of rubrcdoxin impressively demonstrates that there is considerably more detailed structural information contained in the x-ray intensity data than is evident in electron density maps calculated with ;\I I R phases
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