5084 Nucleic Acids Research, 2008, Vol. 36, No. 15 interfaces (8,9). Examples of these approaches include the addition and subsequent removal of puriﬁcation tags; deletions of disordered regions including termini, loops and domains by recombinant techniques or limited proteolysis and replacement of highly entropic residues (e.g. lysines and glutamic acids) by the ‘surface entropy reduction’ method (10). Alterations of proteins to improve crystallization include the substitution of residues known to be involved in crystallization, systematic or random alteration of surface residues to create a library of poten- tially crystallizable proteins, and alteration of known crys- tal contacts that could potentially lead to new crystal forms. HIV-1 RT is a heterodimer consisting of subunits with masses of 66 kDa (p66) and 51 kDa (p51). The two sub- units, p66 and p51, consisting of 560 and 440 residues, respectively, are produced by cleavage of the Gag–Pol polyprotein precursor by HIV-1 protease. They share a common amino terminus. HIV-1 RT crystallizes with dif- ferent space groups and unit cells, and the resulting crys- tals diﬀract X-rays to diﬀerent resolution depending on the nature of the complex (e.g. nucleic acid, NNRTI, etc.) and of the HIV-1 RT itself. Three diﬀerent versions of HIV-1 RT, varying in termini and HIV-1 strain sequence, have been used for crystallization of RT/ NNRTI complexes. Each of the three versions crystallizes with a characteristic space group symmetry: P212121 (11); C2 (12,13) and C2221 (14). To produce crystals of HIV-1 RT/TMC278 complex that diﬀracted to high resolution, we used an iterative high-throughput approach involving multiple rounds of expression, puriﬁcation and crystallization. In each round, the plasmid construct that produced crystals with the highest resolution of X-ray diﬀraction was used as the basis for the next round of mutagenesis. This iterative approach made it possible to develop HIV-1 RTs with better crystallographic characteristics (Figure 1). MATERIAL AND METHODS Expression vector and mutant construction The HIV-1 RT-encoding DNA from the Q258C-RT con- struct (15) was ligation-independent cloned (LIC) into pCDF-2 Ek/LIC with the LIC DuetTM Minimal Adaptor (Novagen, San Diego, CA USA) according to manufacturer’s recommendations. The HIV-1 RT-encod- ing dual expression vector is designated pRT1. Mutagenesis was completed using methylated overlap- extension ligation-independent cloning (MOE-LIC). See Figure 2A for the location and pairing of the primers on pRT1. The methylated and nonmethylated primers are listed in Supplementary Table 3. For mutagenizing ORF-2 (p66), mutagenesis overlap extension PCR was performed using mutated overlap seg- ments with the 20-O-methylated primers to amplify the full insert with PfuUltraTM II Fusion HS DNA Polymerase (Stratagene, La Jolla, CA USA). The vector, with p66 removed to minimize false positives, was ampliﬁed with complementary methylated primers in a separate reaction. The PCR products were then gel puriﬁed, and 0.04 pmols of vector and insert were mixed at a 1 : 1 molar ratio in a buﬀer containing 25 mM Tris pH 8.0, 5 mM MgCl2, Figure 1. Iterative approach to crystal engineering.