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Selenomethionine-labeled protein was prepared by growing the expression strain of Escherichia coli in liquid minimal media with selenomethionine replacing methionine. Labeled protein was purified and crystallized in the same manner as the native transposase, except for the inclusion of 2 mM tris (2-carboxyethyl)-phosphine hydrochloride. A ter(pyridine)PtCl derivative containing two heavy atom sites was produced by soaking crystals in a 0.5 mM solution for 12 hours, and a uranyl acetate derivative containing three heavy-atom sites was produced from a 36-hour soak at 5 mM. Diffraction data were collected at the Structural Biology Center beamline at the Advanced Photon Source in Argonne, IL (Table 1). Data were integrated, scanned and reduced with the HKL software (58). Phases from multiple isomorphous replacement were calculated and improved by density modification using CN5, version 0.5 (59). The model was built into the resulting electron density map with the program TURBO-FRODO (60). Thereafter, the model was improved through cycles of manual model building and refinement by simulated annealing with cross-validated maximum likelihood (61, 62) with CN5, version 1.0.
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We thank L. Mahnke for providing the first samples of purified transposase, J. B. Thoden for help in collecting the x-ray data, and T. Naumann, A. Bhasin, M. Steiniger-White and R. Saecker for helpful discussions. We gratefully acknowledge the help of N. Duke, F. Rotella, and A. Joachimak at the Structural Biology Center Beamline, Argonne National Laboratory, in collecting the data. This research was supported in part by NIH grants AR35186 (I.R.) and CM50692 (W.S.R.). W.S.R. is a recipient of a Vilas Associates Award and is the Evelyn Mercer Professor of Biochemistry and Molecular Biology. D.R.D. was supported by the NIH Biotechnology Training Grant. The use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research, Contract W-31-109-Eng-38. The PDB accession number for the coordinates and structure factors is 1F3I for the transposase/DNA complex.
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