The ﬁrst round of mutagenesis/crystallization produced RT1–RT10 and crystals of the resulting modiﬁed RT/TMC278 complexes that diﬀracted to worse than 10 A˚ resolution (Figure 2D). However, one HIV-1 RT mutant, in which p66 was terminated at residue 555, pro- duced larger crystals than those terminated at residue 560. In the second round, we attempted to optimize the termini for both the p66 and p51 subunits. To avoid possible interference with optimal packing in the crystal lattice as well as to allow for additional packing arrangements, the disordered residues at the termini of both subunits, includ- ing puriﬁcation tags, were removed prior to crystallization. The C-termini were truncated at residue 555 for p66 and 428 for p51 based on the knowledge of less-ordered regions at the termini in published RT crystal structures. Of the three constructs generated in round two, RT13A, which had an N-terminal HRV14 3C cleavable (His)6-tag, gave the highest yield of monodisperse protein [post-(His)6- tag cleavage], as measured by dynamic light scattering (data not shown), and larger crystals (but with no improve- ment in X-ray diﬀraction quality), suggesting this version as the best candidate to be used as the template for the next round of mutants. RT13A was the template for the third round of muta- genesis, resulting in constructs RT21–RT35. The crystals of RT24A/TMC278 complex diﬀracted X-rays to 3.3 A˚ Nucleic Acids Research, 2008, Vol. 36, No. 15 5087 resolution, which was better resolution than had been obtained with any previous version of HIV-1 RT com- plexed with TMC278. The 3.3 A˚ diﬀraction dataset was anisotropic and produced multiple lattices in the diﬀrac- tion patterns; we did not obtain a dataset suitable for structure determination. Up to this point, all HIV-1 RT versions we tested had the p66 Q258C mutation that was used for cross-linking HIV-1 RT to nucleic acid (15,22,23). In the fourth round of mutagenesis, we reverted residue 258 to glutamine to remove any unwanted chemical reac- tivity that might result from having a surface cysteine residue not cross-linked to nucleic acid. New crystal form and high-resolution diffraction from RT52A/NNRTI crystals RT52A (Figure 2E), which is the same as RT24A with the original glutamine at position 258, produced crystals within 1–3 days when complexed with TMC278 and other NNRTIs. The crystals of the RT52A/NNRTI com- plexes diﬀracted X-rays to high resolution (often better then 2.0 A˚ ). The quality of the 1.8 A˚ RT52A/TMC278 structure (6) is evident from the electron density map of the inhibitor shown in Figure 3A. The RT52A/NNRTI complexes represent a new crystal form of HIV-1 RT. This new crystal form has preserved the symmetry of its Figure 3. Crystal Structure of RT52A with TMC278 at 1.8 A˚ resolution. (A) Simulated annealed Fo–Fc omit map (3s contours) for TMC278. (B) Typical 1B1-RT/NNRTI residues involved in crystal packing (pdb code: 1S9E). Residues involved in crystal contacts of HIV-1 RT are shown as space ﬁlled (residues within 4.5 A˚ of the asymmetric unit). (C) RT52A/TMC278 complex residues involved in crystal contacts. (D) Unliganded RT69A residues involved in crystal contacts.