5086 Nucleic Acids Research, 2008, Vol. 36, No. 15 Data collection and structure determination Crystals of RT52A were ﬂash-cooled by immersion into liquid nitrogen after treating the crystals for 2–10 s in a cryoprotective solution containing crystallization well solution plus 27% ethylene glycol and the inhibitor at the same concentration as in the hanging drop. Best results, in terms of sharper diﬀraction spots and signal to noise ratio (I/s), were obtained by using MicroMounts (MiTeGen, Ithaca, NY USA) for mounting the crystals. Crystals were screened and diﬀraction data- sets were collected at the Cornell High Energy Synchrotron Source (CHESS) F1 and A1 beamlines, National Synchrotron Light Source (NSLS) beamlines X25 and X29, and Advanced Photon Source (APS) at Argonne National Laboratory (ANL), SER-CAT beam- line 19ID. The diﬀraction data were indexed, integrated, scaled and merged using HKL2000 (18). The resolution of the data was estimated using the last resolution shell values for completeness, R-merge and the ratio of I to s(I). X-ray diﬀraction data for apo-RT69A was collected at CHESS using the above protocol. The crystal structure of apo-RT69A was solved by molecular replacement using apo-RT structure [PDB ID 1DLO (19)] as the starting model. Cycles of model build- ing guided by high-resolution structures of RT/TMC278 complex and an RT/RNase H-inhibitor complex (Himmel et al., personal communication), solvent modeling and reﬁnement generated the ﬁnal model of apo-RT69A struc- ture that is reﬁned at 1.85 A˚ resolution to Rwork and Rfree of 0.238 and 0.252, respectively. The atomic coordinates and structure factors are deposited in Protein Data Bank (PDB) with accession code 3DLK. RT activity assays The processivity assay used a DNA/DNA template- primer (20). The RNase H activity assay was performed as described (21). Additional methods Other experimental methods, including CD spectroscopy, dynamic light scattering and details of RT enzymatic activity assays are given in Supplementary Methods. RESULTS Coexpression and mutant cloning A coexpression system was used that makes it possible to specify the exact sequence and termini of the two subunits independently (Figure 2A). In the initial coexpression con- struct, the encoded p51 subunit consisted of 428 residues plus a hexahistidine puriﬁcation tag at the C-terminus (15,22,23). This construct also encodes the p66 Q258C mutant, which is used to cross-link nucleic acid to the modiﬁed RT for X-ray crystallographic studies. In Escherichia coli the HIV-1 RT coexpression plasmid pRT1 produces large amounts of HIV-1 RT ( 40 mg/l) under standard conditions. RT mutants were generated using a rapid, high yield and inexpensive mutagenesis system. Donahue et al. (16) described a ligation-independent cloning technique in which terminator primers are used to create 12–15 nt complementary overhangs on the insert and vector. The insert and vector are annealed and transformed into bac- teria, thereby avoiding any post-PCR enzymatic steps. The terminating residue in the primer is a 20-O-methylated nucleotide, which causes the thermostable polymerases Taq or Pfu to terminate DNA synthesis (Figure 2B). There are two major limitations with this technique: (i) the 20-O-methylated primers cost $100 per pair for each insert and (ii) the site of 20-O-methylation has an 20% mutation rate. We combined the terminator primer technique with overlap-extension mutagenesis (24) to develop a rapid mutagenesis protocol for HIV-1 RT called MOE-LIC (see Supplementary Movie). In the MOE-LIC approach, the terminators lie outside the open reading frame (ORF), which avoids problems that could arise from unwanted mutagenesis in the coding or regulatory regions. Overlap-extension PCR makes it possible to generate either novel or mutagenized inserts that can be cloned into the expression plasmid (24,25). For the coexpression system a total of three terminator primer pairs (Figure 2A) was required which cost approximately $300. The termi- nator primer pairs are complementary; there is no com- plementarity between pairs, which allows for eﬃcient cloning and directional speciﬁcity. The technique is cost eﬀective because the same terminator primers were used in all of the cloning. Error rates were found to be extremely low; we found one unintended mutation per 30 mutants produced (1 error in 50 000 nt sequenced). Mutagenesis and crystallization A protein engineering strategy designed to improve the crystallization of HIV-1 RT was developed as follows: (i) disrupt or enhance known common crystal contacts in the existing crystal forms of HIV-1 RT; (ii) remove high B-factor patches, primarily the disordered termini seen in the parent C2 RT/NNRTI crystal form; (iii) reduce surface entropy by converting lysine and glutamic acid patches to alanine (10); (iv) choose amino acids to mutate based on the available information about multiple crystal forms of HIV-1 RT (e.g. sequence variations, dif- ferent sets of crystal contacts, ordered/disordered regions, etc.); (v) avoid mutating conserved residues and (vi) use iterative rounds of mutagenesis/crystallization to improve the quality of X-ray diﬀraction (Figure 1). Figure 2C shows the location of the mutations that were made for the crystallization trials (see Supplementary Table 1 for a complete list of the 59 HIV-1 RT variants and the diﬀrac- tion resolution of the crystals). For the initial crystal screening of each HIV-1 RT variant (Supplementary Table 2), 18 crystallization conditions were chosen from previously reported crystallographic studies of HIV-1 RT (14,17,26,27, Himmel, D.M). Crystallization of individual HIV-1 RT samples was attempted in parallel experiments with unliganded RT, RT complexed with TMC278, and in complexes with other NNRTIs or RNHIs [e.g. b-thujapli- cinol (28)].