Nucleic Acids Research, 2008, Vol. 36, No. 15 5089 Table 1. Comparison of engineered and nonengineered crystal forms Nonengineered RT/NNRTI PDB code:1S9E RT52A/TMC278 RT69A/unliganded Nonengineered RT/unliganded PDB code: 1DLO Thumb conformation Space group Average unit cell parameters Unit cell volume (A3) Molecules/asymmetric unit Vm (A˚ 3/Da) Solvent content (%) Residue pairs in crystal contacts (4.5 A˚ apart) Buried surface area in crystal contacts (A˚ 2) C2 a = 225, b = 69, c = 104 A˚; = 1048 1.57 106 1 3.35 64 97 1556 C2 a = 163, b = 73, c = 110 A˚; = 1008 1.27 106 1 2.77 55 194 2707 C2 a = 236, b = 70, c = 93 A˚; = 1068 1.48 106 1 3.17 61 104 1529 C2 a = 164; b = 72; c = 109; = 1048 1.25 106 1 2.68 54 205 2902 Table 2. Diﬀraction data and reﬁnement statistics Unliganded RT69A PDB ID 3DLK X-ray source CHESS F1 Wavelength (A˚ ) 0.9176 Space group C2 Cell constants (a, b, c in A˚; in degrees) 164.01, 72.04, 109.33; 104.38 Resolution range (A˚ ) (last shell) 50–1.85 (1.88–1.85) Number of unique reﬂections 99 493 (257 025) (number of observations) Completeness (%) (in last shell) 94.5 (84.8) R-merge (in last shell) 0.074 (0.588) Average I/s(I) (in last shell) 14.4 (1.9) Sigma cut-oﬀ |I| < 0.5s(I) Reﬁnement statistics Total number of atoms 8051 (188) (solvent atoms) Resolution (A˚ ) 40.0–1.85 Number of reﬂections (Rfree set) 99 441 (2991) Completeness (%) (minus Rfree set) 94.3 (91.4) Cutoﬀ criteria |F| < 0 Rwork 0.238 Rfree 0.252 Root mean square deviations Bond lengths (A˚ ) 0.006 Bond angles (degrees) 1.313 mutations present in RT52A, also produced improved RNHI-containing crystals. RT97A forms apo-crystals that diﬀract X-rays to 2.1 A˚ resolution. An 1.85 A˚ structure of apo-RT Crystal structure of apo-RT69A is reﬁned at 1.85 A˚ reso- lution to an Rwork and Rfree of 0.238 and 0.252, respec- tively. Like the previously determined apo-RT structures [PDB ID: 1DLO (19), 1HNV (27)], apo-RT69A contains no NNRTI-binding pocket and has the thumb and ﬁngers subdomains in a closed conformation. Superposition of the p51 subunit and the connection-RNase H domains Figure 4. Stereo view of electron density in the RNase H domain of apo-RT69A. Stereo view of the 3Fo–2Fc map (calculated at 1.85 A˚ resolution and contoured at 2.5s) surrounding Tyr532 in the RNase H domain of RT69A. of p66 for apo-RT69A, 1HNV and 1DLO structures results in a RMSD of 1.83 and 1.24 A˚ , respectively, with the major conformational diﬀerences in all three struc- tures between the ﬁngers and thumb subdomains (Supplementary Figure 4). Most of the apo-RT69A struc- ture is well deﬁned by high-resolution electron density (Figure 4). Like the high-resolution RT/NNRTI (TMC278) structure, the high-resolution apo-RT69A structure provides another distinct functional state of RT that can be used in designing new classes of inhibitors. DISCUSSION There is no generalized blueprint for determining the best way to crystallize a protein, and there is no simple proto- col for improving the quality of diﬀraction of protein crystals. We were able to use protein engineering to improve the diﬀraction resolution of a very important HIV-1 drug complex from 6 to 1.8 A˚ ; this result has implications for the design of anti-AIDS drugs and also provides support for the idea that rational approaches