Nucleic Acids Research, 2008, Vol. 36, No. 15 5085 Figure 2. Mutagenesis of RT. (A) Schematic showing the three binding sites (arrows) of the 20-O-methylated primers used in MOE-LIC. Locations of speciﬁc restriction enzyme cut sites are also indicated. (B) Annealed duplex of the primer terminated insert and vector with 20-O-methyl nucleotides indicated by Me. (C) Cartoon of RT color-coded by the p66 subdomains. All mutations made in this study are indicated as spheres. The beneﬁcial mutations are colored yellow and labeled. (D) Flowchart showing the generation of the mutants, which are color-coded to show X-ray diﬀraction resolution of the crystals. Stars denote those mutants for which the unliganded crystals show improved resolution. (E) Diagram of RT1A, RT52A and RT69A. 0.025 mg/ml BSA and 2.5 mM DTT in a 20 ml volume. The mixture was heated to 708C and cooled slowly over 2 h in a water bath. Once cooled to 408C, 1 ml of 25 mM EDTA was added and the mixture incubated at room tempera- ture for 5 min before being desalted using a Centri-Sep column (Princeton Separations, Adelphia, NJ USA) or by ethanol precipitation (16). Desalted annealed DNA of 5 ml was added to electrocompetent NovaBlue cells (Novagen) and electroporated according to manufac- turer’s recommendations. Expression and purification of RT pRT containing BL21-CodonPlus -RIL cells were induced with 1 mM IPTG at an OD600 of 0.9 followed by expression at 378C for 3 h. Ni-NTA puriﬁcation was performed according to the manufacturer’s recommenda- tions (Qiagen, Valencia, CA USA) with the following modiﬁcations: no added lysozyme, 600 mM NaCl in each of the standard buﬀers, 0.1% Triton X-100 added to the lysate and wash buﬀers and a high-salt wash step performed with 1.2 M NaCl added to the standard wash buﬀer. After elution the HRV14 3C protease was added (1 : 100 ratio of protease : RT) and incubated at 48C over- night. Mono Q was performed as described (17). The RT was buﬀer exchanged and concentrated to 20 mg/ml in 10 mM Tris pH 8.0 and 75 mM NaCl. The concentrated RT was aliquoted and stored at 808C or placed at 48C for immediate crystallization. Crystallization Inhibitors were dissolved in dimethyl sulfoxide (DMSO) prior to addition to protein sample. The RT was screened unliganded, with a 2.5-fold molar excess of NNRTI, or with a 5-fold molar excess of RNase H inhibitor (RNHI) using the hanging-drop vapor diﬀusion method. The inhibitor–mutant complex was incubated at room tem- perature for 10 min prior to addition of the crystallization solution. Depending on the number of samples being screened, EasyXtal DG-Tools (Qiagen) or Linbro Plates (Hampton Research, Aliso Viejo, NJ USA) crystallization trays were used for screening. Based on visually identiﬁed crystal hits, further optimization was used to obtain dif- fraction quality crystals. RT52A and RT69A crystals were produced in a matrix of 24 conditions from 9% to 12% PEG 8000, 50 mM imidazole pH 6.0–6.8, 10 mM sper- mine, 15 mM MgSO4 and 100 mM ammonium sulfate. All successful crystallization experiments were performed at 48C.