In addition to improving the opportunities to develop more eﬀective NNRTIs and RNHIs, the ability to produce high- resolution HIV-1 RT crystals quickly and easily should make it possible to use HIV-1 RT in fragment screening assays (29). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We acknowledge personnel at the Cornell High Energy Synchrotron Source (CHESS), Brookhaven National Laboratory (BNL), Advanced Photon Source Argonne National Laboratory (APS) and Liang Tong of Columbia University for support of data collection. Members of our laboratories provided valuable discus- sions and assistance, including Stefan Saraﬁanos, Chhaya Dharia, Chun Chu, Rajiv Bandwar, Thomas Acton, Sergio Martinez and Jason Schifano. E.A. is grate- ful to the National Institutes of Health (Grants AI 27690 MERIT Award and P01 GM 066671) for support of RT structural studies. S.H.H. was supported by the Intramural Research Program of National Institutes of Health, National Cancer Institute, Center for Cancer Research and National Institute of General Medical Sciences. Funding to pay Open Access publication charges for this article was provided by the NIH. Conﬂict of Interest statement. None declared. REFERENCES 1. Coﬃn,J.M. (1995) HIV population dynamics in vivo-implications for genetic variation, pathogenesis, and therapy. Science, 267, 483–489. 2. Larder,B.A. and Kemp,S.D. (1989) Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science, 246, 1155–1158. 3. Janssen,P.A., Lewi,P.J., Arnold,E., Daeyaert,F., de Jonge,M., Heeres,J., Koymans,L., Vinkers,M., Guillemont,J., Pasquier,E. et al. (2005) In search of a novel anti-HIV drug: multidisciplinary coordination in the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2, 6-dimethylphenyl]amino]-2- pyrimidinyl]amino]benzonitrile (R278474, rilpivirine). J. Med. Chem., 48, 1901–1909. 4. Geretti,A.M., (2008) Shifting paradigms: the resistance proﬁle of etravirine. J. Antimicrob. Chemother. [Epub ahead of print; doi:10.1093/jac/dkn248]; June 19, 2008. 5. Das,K., Clark,A.D. Jr, Lewi,P.J., Heeres,J., de Jonge,M.R., Koymans,L.M., Vinkers,H.M., Daeyaert,F., Ludovici,D.W., Kukla,M.J. et al. (2004) Roles of conformational and positional adaptability in structure-based design of TMC125-R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and eﬀective against wild-type and drug-resistant HIV-1 variants. J. Med. Chem, 47, 2550–2560. 6. Das,K., Bauman,J.D., Clark,A.D. Jr., Frenkel,Y.V., Lewi,P.J., Shatkin,A.J., Hughes,S.H. and Arnold,E. (2008) High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic ﬂexibility explains potency against resistance mutations. Proc. Natl Acad. Sci. USA, 105, 1466–1471. 7. Das,K., Lewi,P.J., Hughes,S.H. and Arnold,E. (2005) Crystallography and the design of anti-AIDS drugs: conformational ﬂexibility and positional adaptability are important in the design of non-nucleoside HIV-1 reverse transcriptase inhibitors. Prog. Biophys. Mol. Biol, 88, 209–231. Nucleic Acids Research, 2008, Vol. 36, No. 15 5091 8. Dale,G.E., Oefner,C. and D’Arcy,A. (2003) The protein as a variable in protein crystallization. J. Struct. Biol, 142, 88–97. 9. Derewenda,Z.S. (2004) The use of recombinant methods and molecular engineering in protein crystallization. Methods, 34, 354–363. 10. Derewenda,Z.S. and Vekilov,P.G. (2006) Entropy and surface engineering in protein crystallization. Acta Crystallogr. D Biol. Crystallogr, 62, 116–124. 11. Ren,J., Esnouf,R., Garman,E., Somers,D., Ross,C., Kirby,I., Keeling,J., Darby,G., Jones,Y., Stuart,D. et al. (1995) High reso- lution structures of HIV-1 RT from four RT-inhibitor complexes. Nat. Struct. Biol., 2, 293–302. 12. Kohlstaedt,L.A., Wang,J., Friedman,J.M., Rice,P.A. and Steitz,T.A. (1992) Crystal structure at 3.5 A˚ resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science, 256, 1783–90. 13. Ding,J., Das,K., Tantillo,C., Zhang,W., Clark,A.D. Jr., Jessen,S., Lu,X., Hsiou,Y., Jacobo-Molina,A., Andries,K. et al. (1995) Structure of HIV-1 reverse transcriptase in a complex with the non- nucleoside inhibitor alpha-APA R 95845 at 2.8 resolution. Structure, 3, 365–379. 14. Hogberg,M., Sahlberg,C., Engelhardt,P., Noreen,R., Kangasmetsa,J., Johansson,N.G., Oberg,B., Vrang,L., Zhang,H., Sahlberg,B.L. et al. (1999) Urea-PETT compounds as a new class of HIV-1 reverse transcriptase inhibitors. 3. Synthesis and further structure-activity relationship studies of PETT analogues. J. Med. Chem., 42, 4150–4160. 15. Saraﬁanos,S.G., Clark,A.D. Jr., Tuske,S., Squire,C.J., Das,K., Sheng,D., Ilankumaran,P., Ramesha,A.R., Kroth,H., Sayer,J.M. et al. (2003) Trapping HIV-1 reverse transcriptase before and after translocation on DNA. J. Biol. Chem., 278, 16280–16288. 16. Donahue,W.F., Turczyk,B.M. and Jarrell,K.A. (2002) Rapid gene cloning using terminator primers and modular vectors. Nucleic Acids Res., 30, e95. 17. Clark,A.D. Jr., Jacobo-Molina,A., Clark,P., Hughes,S.H. and Arnold,E. (1995) Crystallization of human immunodeﬁciency virus type 1 reverse transcriptase with and without nucleic acid sub- strates, inhibitors, and an antibody Fab fragment. Methods Enzymol., 262, 171–185. 18. Otwinowski,Z. and Minor,W. (1997) Processing of X-ray Diﬀraction Data Collected in Oscillation Mode. Methods Enzymol., 276, 307–326. 19. Hsiou,Y., Ding,J., Das,K., Clark,A.D. Jr., Hughes,S.H. and Arnold,E. (1996) Structure of unliganded HIV-1 reverse transcrip- tase at 2.7 A˚ resolution: implications of conformational changes for polymerization and inhibition mechanisms. Structure, 4, 853–860. 20. Boyer,P.L., Saraﬁanos,S.G., Arnold,E. and Hughes,S.H. (2002) The M184V mutation reduces the selective excision of zidovudine 50-monophosphate (AZTMP) by the reverse transcriptase of human immunodeﬁciency virus type 1. J. Virol, 76, 3248–3256. 21. Boyer,P.L., Stenbak,C.R., Clark,P.K., Linial,M.L. and Hughes,S.H. (2004) Characterization of the polymerase and RNase H activities of human foamy virus reverse transcriptase. J. Virol, 78, 6112–6121. 22. Huang,H., Chopra,R., Verdine,G.L. and Harrison,S.C. (1998) Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science, 282, 1669–1675. 23. Huang,H., Harrison,S.C. and Verdine,G.L. (2000) Trapping of a catalytic HIV reverse transcriptase template:primer complex through a disulﬁde bond. Chem. Biol., 7, 355–364. 24. Ho,S.N., Hunt,H.D., Horton,R.M., Pullen,J.K. and Pease,L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77, 51–59. 25. Horton,R.M., Hunt,H.D., Ho,S.N., Pullen,J.K. and Pease,L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 77, 61–68. 26. Chan,J.H., Hong,J.S., Hunter,R.N., Orr,G.F., Cowan,J.R. 3 rd, Sherman,D.B., Sparks,S.M., Reitter,B.E., Andrews,C.W., Hazen,R.J. et al. (2001) 2-Amino-6-arylsulfonylbenzonitriles as non- nucleoside reverse transcriptase inhibitors of HIV-1. J. Med. Chem, 44, 1866–1882. 27. Rodgers,D.W., Gamblin,S.J., Harris,B.A., Ray,S., Culp,J.S., Hellmig,B., Woolf,D.J., Debouck,C. and Harrison,S.C. (1995) The structure of unliganded reverse transcriptase from the human