TABLE 2.
Structural statistics of the 20 best NMR structures of copsin
Structure calculation by simulated annealing protocol of CYANA (15) after energy minimization using Amber (16) is shown. A, Structure calculation by simulated annealing protocol of CYANA (15) without disulfide constraints. B, Structure calculation by simulated annealing protocol of CYANA (15) with defined ensemble of disulfide bonds. C, Structure calculation parameters after energy minimization using Amber (16).
| Aa | Bb | Cc | |
|---|---|---|---|
| NMR restraints | |||
| Distance restraints | 809 | 823 | 823 |
| Intraresidual and sequential (|i − j| = 1) | 462 | 459 | 459 |
| Medium range (1 < |i − j| < 5) | 115 | 126 | 126 |
| Long range (|i − j|> = 5) | 232 | 230 | 230 |
| Hydrogen bondsd | 8 | 8 | 8 |
| Torsion angles | 0 | 0 | 0 |
| Disulfide bonds | 0 | 6 | 6 |
| Energy statisticse,f | |||
| Average distance constraint violations | 0 | 0 | |
| 0.1–0.2 Å | 8.8 ± 2.0 | ||
| 0.2–0.3 Å | 2.2 ± 1.2 | ||
| 0.3–0.4 Å | 0.2 ± 0.4 | ||
| >0.4 Å | 0.0 ± 0.0 | ||
| Maximal (Å) | 0.28 ± 0.03 | ||
| Average angle constraint violations | 1 | 1 | 0 |
| <5° | 0.0 ± 0.0 | ||
| >5° | 0.0 ± 0.0 | ||
| Maximal (°) | 0.0 ± 0.0 | ||
| Mean AMBER violation energy | |||
| Constraint (kcal mol−1) | 9.1 ±1.2 | ||
| Distance (kcal mol−1) | 9.1 ±1.2 | ||
| Torsion (kcal mol−1) | 0.0 ± 0.0 | ||
| Mean AMBER energy (kcal mol−1) | −2331.2 ± 5.2 | ||
| Mean deviation from ideal covalent geometry | |||
| Bond length (Å) | 0.0043 ± 0.0001 | ||
| Bond angle (°) | 1.728 ± 0.009 | ||
| Ramachandran plot statisticse,f,g | |||
| Residues in most favored regions (%) | 70.4 | 64.2 | 79.2 ± 1.9 |
| Residues in additionally allowed regions (%) | 29.6 | 35.8 | 20.7 ± 2.1 |
| Residues in generously allowed regions (%) | 0.1 | 0.0 | 0.1 ± 0.5 |
| Residues in disallowed regions (%) | 0.0 | 0.0 | 0.0 ± 0.0 |
| Root mean square deviation to mean structuree,f | |||
| Backbone atoms (Å) | 0.50 ± 0.09 | 0.43 ± 0.09 | 0.34 ± 0.09 |
| Heavy atoms (Å) | 0.98 ± 0.16 | 0.87 ± 0.11 | 0.78 ± 0.08 |
| Target function | 0.21 ±0.003 | 0.14 ±0.003 | |
a No constraint for disulfide linkage was used.
b Each disulfide bond was explicitly defined by upper and lower distance limit restraints between the sulfur and carbon atoms of the two linked cysteines: Cys3–Cys32, Cys10–Cys40, Cys18–Cys48, Cys22–Cys50, Cys25–Cys57, and Cys35–Cys54.
c 20 structures of column B with the lowest target function were energy minimized using Amber (16).
d H-bond constraints were identified from HNCO (14) experiments and slow exchanging amide protons in D2O.
e Statistics computed for the best 20 structures (see column C).
f Based on structured residue range, residues 3–57.
g Ramachandran plot, as defined by the program Procheck (48).