REFERENCES AND NOTES

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  18. Recombinant IFN-g D' was expressed in E. coli by procedures similar to those previously described by Tanaka et al(11). It was purified to constant specific antiviral activity and apparent homogeneity with SDS-polyacrylamide gel electrophoresis by using standard chromatographic procedures. [H.V.Le, C.A.Mays, R.Syto, T.L.Nagabhushan, P.P.Trotta, in The Biology of the Inteferon System 1985, W. E. Stewart II and H. Schellekens, Eds. (Elsevier, Amsterdam, 1985), pp. 73-80].
  19. Crystals for x-ray diffraction studies were grown from 40% saturated ammonium sulfate solutions at pH 5.9 (17). The native data was obtained in part by using crystals grown under microgravity conditions on space shuttle mission STS-26 (20). The space group is R32 with hexagonal cell parameters a = b = 114.0 A and c = 315.0 A. The corresponding rhombohedral axes are a = 123.9 A and a = 54.8°. There are two dimers in the asymmetric unit, and the solvent volume fraction is 60%.
  20. L.J. DeLucas et al., Science 246, 651 (1989).
  21. Potential heavy-atom derivatives were prepared by soaking crystals for 24 hours at 22°C in 65% ammonium sulfate solutions that contained 0.05 M MES, pH 5.9. Data for native crystals and the heavy-atom derivatives were collected by using CuKa radiation from a Rigaku RU-300 rotating anode x-ray source operating at 40 kV and 100 mA. Oscillation frames covered 0.25° and were measured for 5 min. In order to obtain complete data sets with multiple measurements of all reflections, multiple crystals were often used. Indexing and integration of intensity data were carried out with the XENGEN processing programs [A.J.Howard et al., J. Appl. Crystallogr. 20, 383 (1987)].
  22. The derivatives are divided into two groups based on the site of heavy-atom binding. The first group consists of K2Pt(NO2)4, K2PtC14, and Na2IrCl6. Each of these derivatives displays one binding site per subunit with the four binding sites related by 222 noncrystallographic symmetry. The heavy-atom sites for these derivatives are essentially the same. The derivatives show some variation in quality, with the K2Pt(NO2)4 derivative being slightly better than any of the other three. The second group of heavy-atom derivatives consists of KAu(CN)2, K2Pt(CN)4 and K2HgI4. As was the case for the first group of derivatives, each subunit contains one binding site, with the four resulting sites related by noncrystallographic symmetry. The 1 mM KAu(CN)2 derivative has one site that appears to have much greater affinity than the other three sites within the crystallographic tetramer.
  23. B.C. Wang, Methods Enzymol. 115, 90 (1985).
  24. 24. One of the four polypeptide chains was traced from this map and then aligned with the amino acid sequence of the portions of the chain where the electron density was well defined. A starting model including all main-chain and side-chain atoms was built automatically from the initial Ca positions with a modified version of the fragment-fitting routines of T. A. Jones and S. Thirup [EMBO J. 5, 819 (1986)]. The other three polypeptides were generated by using the noncrystallographic symmetry. Each residue was then fit to the unaveraged electron density with the aid of a local version of the graphics program TOM [C.Cambillau and E.Horjales, J.Mol.Graphics 5,174 (1987)], insuring that each side-chain and main-chain dihedral angle had a reasonable value.
  25. A.T. Briinger, J. Kuriyan, M. Karplus, Science 235, 458 (1987).
  26. 26. The crystal packing mimics the cubic space group F432 as a result of the arrangement of the noncrystallographic twofold axes. The coordinates of the atoms in the pseudocubic cell are related to those in the rhombohedral cell by the following transformation matrix:
    DRAWTHEMATRIX
    where the translation vector (1/4,1/4,1/4) reflects the difference in origin definition between R32 and F432. When this transformation matrix is applied to the center of the four subunits, one obtains approximately the 222 special position in the cubic space group. If the space group had been F432, there would have been one subunit per asymmetric unit.
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  44. Abbreviations for the amino acid residues are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; 1, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  45. The alpha-carbon coordinates for recombinant human IFN-g D' will be deposited with the Brookhaven Protein Data Bank. We thank S.K. Narula and staff for the expression of recombinant human IFN-g D' in Esherichia coli, G.F. Seelig and P. Reichert for preparation of purified protein, S. Senadhi for preparation of recombinant human IFN-g D' crystals, and C. Smith and B. Cole for assistance with data processing. Supported by NIH grant CA-13148 and NASA grant NAGW813.

19 October 1990; accepted 20 February 1991

S.E. Ealick(*), Department of Pharmacology, Center for Macromolecular Crystallography and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294.
[Steve has moved to Cornell]

W.J. Cook, Department of Pathology, Center for Macromolecular Crystallography and Comprehensive Cancer Center, University of Alabama at Birmingharn, Birmingham, AL 35294.

S. Vijay-Kumar and M. Carson, Center for Macromolecular Crystallography, University of Alabama at Birmingham, Birminlzham, Al 35294.
[Vijay has moved to Temple]

T.L. Nagabhushan and P.P. Trotta, Schering-Plough Research, Bloomfield, NJ 07003.

C.E. Bugg, Department of Biochemistry, Center for Macromolecular Crystallography and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294.
[Charlie has moved to BioCryst]

*To whom correspondence should be addressed.