5088 Nucleic Acids Research, 2008, Vol. 36, No. 15 parent crystal space group C2 but has distinctly diﬀerent unit cell parameters and crystal contacts (Figure 3B–D). Tighter packing of RT52A molecules in the crystal is evident from a 14% decrease in solvent content and a 19% decrease in unit cell volume compared to NNRTI (Janssen-R129385) complexed with the form of HIV-1 RT we used previously (expression construct designated 1B1) (17). There are nearly twice as many residues involved in crystal packing (within 4.5 A˚ of each other), 194 residues in RT52A/TMC278 structure compared with 97 in the 1B1 RT/R129385 structure. The surface area involved in crystal contacts is increased from 1556 A˚ 2 in the IB1 RT/R129385 structure to 2707 A˚ 2 in the RT52A/ TMC278 structure, calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Fragment screening with RT52A/TMC278 crystals Drug fragment cocktail screening (29,30) is a potentially powerful technique for ﬁnding new inhibitors and new sites for inhibitors to bind, but this approach was diﬃcult with the earlier, moderate resolution crystals of HIV-1 RT. Drug fragment cocktails are usually dissolved in DMSO and soaked into preformed crystals of the target protein in the crystallization solution plus DMSO. To determine if RT52A/TMC278 crystals could be used for fragment cocktail screening, crystals were soaked in 10–20% DMSO before and during cryoprotection. No loss in dif- fraction quality was found with 10% DMSO, and there was only a moderate decline in diﬀraction quality when 20% DMSO was used (2.0 versus 1.8 A˚ , data not shown). The DMSO-soaked crystals were also isomorphous to the ori- ginal RT52A/TMC278 crystals. These results indicate that RT52A/TMC278 crystals are suitable for screening for binding of drug-like molecules and small chemical frag- ments and for lead optimization at both existing and novel-binding sites. The high-resolution HIV-1 RT crystals enable the acquisition of fast and reliable structures and will be critical in structure-based lead optimization. Validation of RT52A and its derivatives Comparison of the RT52A/TMC278 structure with 1B1 RT/NNRTI structures showed that the overall RT fold, distribution of secondary structure elements, and mode of NNRTI binding (6) are very similar, suggesting that the crystal engineering mutations had no signiﬁcant impact on the structure of RT. To test for possible functional eﬀects, proteins RT35A (RT52A without the K172A/K173A mutation), RT51A (RT52A + L100I/K103N), RT52A and RT55A (RT52A + K103N/Y181C) were assayed for DNA-dependent DNA polymerase activity and processiv- ity and for RNase H activity (20,21). Supplementary Figure 2A shows that RT52A has processivity that is simi- lar to wild-type HIV-1 RT (in this assay the wild-type HIV-1 RT was produced by coexpressing p66 with HIV- 1 protease). RT51A has diminished processivity and RT55A an apparent increase in processivity. Each of the mutants has similar RNase H activity and speciﬁcity (Supplementary Figure 2B). For 1B1-RT/NNRTI crystallization studies, only the p66 form of HIV-1 RT was expressed. A p51-like chain was produced via cleavage at residue 447 by a copurifying bacterial protease (Boyer, P.L.), ultimately yielding p66/p51 heterodimer (17). Altering RT52A at the C-terminus of p51 to produce a version that terminates at 447, instead of 428, changed the crystal unit cell to that seen with 1B1-RT complexed with NNRTIs, but with a signiﬁcant reduction in X-ray diﬀraction resolution to 2.7 A˚ (Supplementary Table 1). Additional mutants were constructed to test the contribution of each of the changes in RT52A (Figure 2E), and each of the changes was found to be required for X-ray diﬀraction at high resolution (Supplementary Table 1). Engineering of high-resolution apo-RT crystals Multiple conformations of proteins in a crystal can limit the ability of the crystals to diﬀract X-rays to a high reso- lution, a problem that is particularly acute for a ﬂexible protein like HIV-1 RT. Complexes of HIV-1 RT bound to inhibitors, antibodies, or substrates that may favor a single conformation or a subset of conformations have been used to reduce the ﬂexibility of HIV-1 RT. While RT52A successfully produced crystals of RT/NNRTI complexes that diﬀracted to high resolution, the unli- ganded RT52A crystals diﬀracted to only 3 A˚ resolution (Supplementary Table 1). The apo-form of 1B1 RT (19) crystallizes with diﬀerent unit cell parameters than the 1B1 RT/NNRTI complexes (Table 1). The diﬀerence in the unit cell between unliganded 1B1 RT and the 1B1 RT/ NNRTI crystals is a consequence of packing of two struc- turally distinct (thumb up versus down) conformations of RT. This may explain why RT52A, which was optimized to produce HIV-1 RT/NNRTI crystals diﬀracting to high resolution, failed to produce crystals that diﬀract to high- resolution when crystallized without an NNRTI. A diﬀer- ent set of mutations may therefore be necessary to obtain a high-resolution apo-RT crystal form. Subsequent rounds of mutagenesis focused on obtaining high-resolution crystals of apo-RT and HIV-1 RT com- plexes with RNHI-bound or DNA-bound RT. RT69A, which contains the mutation F160S, produced crystals of apo and RNHI-bound RT that diﬀracted X-rays to 1.8 A˚ resolution (Table 2). Crystals of unliganded RT69A con- tain a unit cell similar to the unit cell of NNRTI bound RT52A but quite distinct from other unliganded struc- tures (Table 1). F160S is located adjacent to the binding cleft for nucleic acid and causes a 1.5 A˚ shift in Y115, which interacts directly with incoming nucleotides during polymerization. RT69A has wild-type levels of RNase H activity, indicating that the enzyme binds nucleic acid eﬃ- ciently and that the RNase H active site is unaﬀected; however, RT69A has reduced processivity, indicating that there may be a reduction in polymerase activity or reduced ability to remain bound to the nucleic acid during DNA synthesis (Supplementary Figure 2C–D). Conse- quently, RT69A may not be the optimal form of HIV-1 RT for studies that involve nucleic acid or studies in which the region near the polymerase active site is important; however, it is suitable for structural studies of RT in complexes with RNHIs (Figure 4). RT97A, which con- tains the mutations P468T/N471D in addition to the