Table 1

Summary of x-ray crystal structure determination. Crystals of pentalenene synthase form as short hexagonal rods and belong to space groupP6 3 with hexagonal unit cell dimensions of a = b = 179.8 Å, c = 56.6 Å; two 38-kD monomers reside in the asymmetric unit and are related by two-fold noncrystallographic symmetry (NCS) (7). Diffraction data from pentalenene synthase crystals were collected at room temperature on an R-AXIS IIc image plate area detector, and intensity data integration and reduction were performed with MOSFLM (19) and CCP4 (20), respectively. For phase determination by multiple isomorphous replacement (MIR), initial heavy atom positions were determined in difference Patterson maps and refined with the program MLPHARE (20, 21). The model was fit into an electron density map calculated with solvent-flipped and NCS-averaged MIR phases extended to 3.3 Å resolution with SOLOMON (20, 22). Subsequent refinement and rebuilding of the native model was performed with X-PLOR (23) and O (24), respectively. Group B factors (one main chain and one side chain B factor per residue) were refined and a bulk solvent correction was applied. Strict NCS constraints were maintained as judged by R free; refinement at 3.3 Å resolution converged to a crystallographic R factor of 0.215 (R free = 0.277). A crystal derivatized with 1.0 mM trimethyllead acetate diffracted to 2.6 Å resolution when flash-frozen at the Cornell High Energy Synchrotron Source (CHESS, beamline A-1, λ = 0.91 Å). Although this crystal was nonisomorphous with native crystals (at room temperature), the 3.3 Å resolution model of pentalenene synthase served as the starting point for rigid-body refinement, followed by iterative rounds of simulated annealing refinement and rebuilding against the 2.6 Å resolution data with X-PLOR (23) and O (24), respectively. Restrained individual B factors were refined and a bulk solvent correction was applied. The quality of the model was improved in the final stages of refinement by releasing the NCS constraints into appropriately weighted restraints as judged byR free. Refinement converged smoothly to a final crystallographic R factor of 0.198 (R free = 0.273). Disordered segments in the final model include Pro2-Gln3, Phe158-Asp164, and Arg314-His337 at the COOH-terminus. The final model has excellent stereochemistry with no residues adopting unfavorable backbone conformations. Since there are no significant structural differences between the 3.3 Å resolution and the 2.6 Å resolution models, and since the lead binding site is removed from the active site (lead makes an interlattice contact between two protein molecules), active site features of the 2.6 Å resolution structure represent those of the native enzyme.

Data collection
Resolution (Å)
Total (N)3808670683194601873441827947065309287
Unique (N)1419728128120931183113848427236714057
Completeness (%)89.887.591.589.096.793.080.286.7
Number of sites1551112
R sym
Outer shell0.2450.1580.2310.4490.5810.1160.1400.272
Phasing (15 − 3.5 Å)
R iso 0.2320.1850.3720.1680.2550.210
Phasing power§
Figure of merit0.506
Resolution (Å)20 − 3.320 − 2.6
Protein atoms (N)21772425
Solvent atoms (N)033
work (N)1249825158
free (N)6361342
R cryst | 0.2150.198
R free | 0.2770.273
rms deviations
Bonds (Å)0.0140.016
Angles (°)1.71.8
  • * TMLA, trimethyllead acetate; PHMPS,p-hydroxymercuri(II)phenylsulfonate; TERPT, chloro(2,2′:6′,2"-terpyridine)platinum(II).

  • Rsym= Σ∥i − 〈Ii〉|/Σ|〈Iil, where Ii is the intensity measurement for reflection i, and 〈Ii〉 is the mean intensity calculated for reflection i from replicate data.

  • Riso = Σ∥FPH| − |FP∥/Σ|FP|, where FPH and FP are the derivative and native structure factors, respectively.

  • § Phasing power = 〈F H〉/E where 〈F H〉 is the root-mean-square heavy-atom structure factor and E is the residual lack of closure error.

  • | R = Σ∥Fo| − |Fc∥/Σ|Fo|, where Rcryst and Rfreeare calculated with the working and test reflection sets, respectively. The test set reflections were held aside throughout refinement.