Table 1

Summary of data collection and refinement statistics. Crystals (space group C2: a = 128.8 Å,b = 68.9 Å, c = 112.0 Å, β = 124.6°) were flash-frozen by being transferred directly from the crystal mother liquor to a stream of boiled-off nitrogen (24). Because crystals of the mutant L90C diffracted significantly better than wild-type protein crystals, the former were used for native data collection. Data were collected from multiple crystals, and six sets were selected and merged to form the native data set used for structure determination. Mercury derivatives were obtained by direct addition of methyl mercury to the crystallization solution of cysteine mutant crystals. MALDI-TOF mass spectrometry confirmed 60 to 90% derivatization of crystals prior to data collection. All data were collected at Cornell High Energy Synchrotron Source (CHESS), station A1, with the Princeton 2K CCD (25). Data were processed with DENZO and SCALEPACK (26) and the CCP4 package (27). Heavy atom positions were determined with SHELX-97 (28) and cross-difference Fourier analysis. These positions confirmed the fourfold noncrystallographic symmetry observed in the self-rotation Patterson function and allowed the determination of initial orientation matrices. An initial model (90% complete) was built into a solvent flattened (64% solvent content), fourfold averaged electron density map with the program O (29). The tracing of the model was facilitated by the use of the mercury atom positions as residue markers. L86C was used solely for this purpose. After torsional refinement (with strict fourfold noncrystallographic symmetry constraints) with X-PLOR 3.851 (30), this model was used in the anisotropic scaling [sharpening (31)] of the native data with X-PLOR. The structure factor sigma values were also rescaled appropriately, and the corrected data were used for all subsequent procedures. Fourfold averaging, solvent flattening, and phase extension were applied in DM (32), resulting in a marked improvement of the electron density that allowed correction of the model and the building of additional residues. Refinement consisted of rounds of positional (in the initial stages phase information was also included as a restraint) and grouped B-factor refinement in X-PLOR. Fourfold noncrystallographic symmetry was highly restrained with the force constant for positional restraints set as 1000 kcal/mol/Å2. The diffuse ion cloud described in the text was initially modeled as one or more K+ ions and several water molecules; however, the results were unsatisfactory. Therefore, this and other strong unmodelled density present in solvent-flattened maps (no averaging included) was Fourier back-transformed, scaled, and included in the refinement procedure as partial structure factors. The final model includes amino acids 23 to 119 of each chain. The following residues were truncated: Arg27 to Cβ, Ile60to Cγ, Arg64 to Cβ, Glu71 to Cβ, and Arg117 to Nɛ. The stereochemistry is strongly restrained, with no outliers on the Ramachandran plot. The highB-factor values reflect the intensity decay of the data beyond 4 Å.

Data collection and phasing
Data setResolution (Å)RedundancyCompleteness overall/outer (%)Rmerge*Phasing powerR-Cullis
Outer Shell3.3–3.22.366.6%0.2863.950
Anisotropic correction
Before sharpeningAverage F.O.M|(30.0–3.2 Å)Average F.O.M| (3.4–3.2 Å)
After sharpening
0.76 0.83 0.55 0.64
Refinement Root-mean-square deviation of
Resolution:10.0–3.2 ÅBond angles:1.096°
R-cryst.§:28.0%Bond lengths:0.005 Å
R-free§:29.0%Ncs related atoms:0.006 Å
No. of reflections with |F|/σ|F| > 2:12054B-factor for ncs related atoms:10 Å2
No. of protein atoms:710 per subunitB-factor for non-bonded atoms:36 Å2
No. of ligand atoms:1 water, 3 K+ions
Mean B-factor for main-chain atoms:90 Å2
Mean B-factor for side-chain atoms:110 Å2
I/σI% of measured
data with I/σI > 2
  • * Rmerge = ΣΣ|I−Ij|/ΣI.

  • Phasing power = 〈|F h|〉/〈E〉.

  • R-Cullis = Σ∥Fph ±Fp|−|Fhc∥/Σ|Fph± Fp|, only for centric data.

  • § R-cryst. = Σ|FpFp(calc)|/Σ|Fp|,R-free the same as R-cryst, but calculated on 10% of data selected in thin resolution shells and excluded from refinement.

  • | Figure of merit.

  • In both cases, fourfold averaging and solvent flattening were applied; Ijis the observed intensity, I is the average intensity,F h is the root-mean-square heavy-atom structure factor, E is the lack of closure error,F ph is the structure factor for the derivative,F p is the structure factor for the native,F hc is the calculated structure factor for the heavy atom, and F p(calc) is the calculated native structure factor. {tt}