5090 Nucleic Acids Research, 2008, Vol. 36, No. 15 can be used to enhance the diﬀraction quality of macro- molecular crystals. Further mutagenesis showed that the terminal truncations primarily deﬁne the unit cell of the RT52A/NNRTI complexes and that the other mutations increase the resolution of the X-ray diﬀraction by stabiliz- ing the crystallized conformation of RT (Figure 5 and Supplementary Table 1). The stabilization of a particular crystallized conformation within the conﬁnes of tighter crystal packing appears to be responsible for the improved diﬀraction (Figure 5). However, the improved resolution is not apparent from the thermal stability measurements by circular dichroism (Supplementary Figure 3). Comparison of X-ray diﬀraction resolution versus Matthews coeﬃcients (A3/Da) of crystal structures of var- ious HIV-1 RT forms indicates that there is an increase in resolution as solvent content (Matthews coeﬃcients) decreases (Figure 5). The highest resolution diﬀraction previously seen was the 2.2 A˚ structure of RT/nevirapine (11), which was obtained by dehydrating the crystals (31). Apparently, dehydrating the crystals reduced the solvent content without fully optimizing the internal stability of the protein molecules in the dehydrated crystal lattice. We introduced mutations on the surface of HIV-1 RT that enhance the stability of RT molecules in the new crystal lattice. The addition of NNRTI-resistance mutations to RT52A/TMC78 crystals caused a decrease in X-ray dif- fraction resolution (2.9 A˚ for L100I/K103N and 2.1 A˚ for K103N/Y181C). The L100I/K103N double mutant increases the EC50 of TMC278 from 0.4 to 8.0 nM while the K103N/Y181C double mutant’s EC50 is 1.0 nM (3). The structures of RT/TMC278 complexes have revealed diﬀerences in the mode of binding for TMC278 with the NNRTI-resistance wild-type and mutant RTs (6). We have shown that the change in pocket conformation is accompanied by changes in the overall conformation of RT (5). Apparently, the L100I/ K103N mutated RT52A, which has a large change in the pocket conformation and in the mode of TMC278 bind- ing, is not as optimized in the crystal lattice as the RT52A/ TMC278 structure, leading to signiﬁcant drop in resolu- tion. Because apo-RT and RT in a complex with NNRTI crystallize diﬀerently and make distinct crystal contacts, two distinct sets of mutations are required to optimize two distinct conformations of RT in the two diﬀerent crystal forms (NNRTI-bound and apo). Iterative protein engineering for crystallization can be applied to other proteins of interest, including other drug targets. Based on our results, the protein of interest should be modiﬁed to remove unstructured residues based on crys- tallographic and partial proteolysis results. Further muta- genesis, based on principles outlined in this article, can be used to improve crystal contacts and reduce the conforma- tional ﬂexibility of the protein. Although the eﬀects of any one set of mutations are diﬃcult to predict, taking a parallel approach that involves iterative steps can be used to improve the X-ray diﬀraction quality of the crystals. We were successful in our initial goal of ﬁnding an HIV-1 RT mutant that gave diﬀraction quality crystals in a complex with TMC278 (6). This success, and in particular, the considerable decrease in the time it takes to grow good crystals, demonstrates the feasibility of high-throughput crystallization of HIV-1 RT in complexes with NNRTIs. Figure 5. Comparison of unit cell and X-ray diﬀraction resolution of mutants. Plot of unit cell (Matthews coeﬃcient) and X-ray diﬀraction resolution (A˚ ) of the mutants that produced crystals that diﬀracted X-rays to better than 4 A˚ resolution. The legend of the table indicates the mutations and the parental template for each of the mutants. RT69A and RT97A are plotted based on crystals with RNHIs bound; all others were complexed with NNRTIs. RT35A is highlighted in bold and RT52A and RT69A are boxed for emphasis.