Table 2. Details of SAXS data collection and analysis.
| α-DsbA2ΔN | FL α-DsbA2 | |
|---|---|---|
| Data-collection parameters | ||
| Instrument | SAXS/WAXS (Australian Synchrotron) | |
| Beam geometry | Point | |
| Wavelength (Å) | 1.033 | 1.127 |
| Sample-to-detector distance (m) | 1.575 | 1.575 |
| q-range (Å−1) | 0.01–0.57 | 0.01–0.48 |
| Exposure time (s) | 16 (8 × 2 s exposures) | 40 (8 × 5 s exposures) |
| Configuation | Concentration series from 96-well plate | WTC-030S5 SEC–SAXS column: flow, 0.5 ml min; inject, 100 µl, 19.8 mg ml−1 |
| Protein concentration range (mg ml−1) | 1.25–5.00 | 0.35–2.20 |
| Temperature (K) | 283 | 293 |
| Absolute intensity calibration | Water | |
| Sample details | ||
| Extinction coefficient [A 280, 0.1%(w/v)] | 0.494 | 0.762 |
| Partial specific volume (cm3 g−1) | 0.735 | 0.735 |
| Contrast, Δρ (1010 cm−2) | 2.88 | 2.89 |
| Molecular mass (from sequence) (kDa) | 21.1 | 27.1 |
| Protein concentration† (mg ml−1) | 1.25 | 0.75 |
| Structural parameters | ||
| I(0) (from Guinier) (cm−1) | 0.0215 ± 0.0001 | 0.04846 ± 0.00003 |
| R g (from Guinier) (Å) | 18.7 ± 0.1 | 28.0 ± 0.1 |
| I(0) [from p(r)] (cm−1) | 0.0215 ± 0.0001 | 0.04845 ± 0.00002 |
| R g [from p(r)] (Å) | 18.8 ± 0.1 | 27.9 ± 0.1 |
| D max (Å) | 63 ± 3 | 87 ± 3 |
| Porod volume (Å3) | 27500 ± 1500 | 91300 ± 4500 |
| Molecular-mass determination | ||
| Molecular mass [from I(0)] (kDa) | 23 ± 1 | 87 ± 5 |
| Molecular mass (from Porod) (kDa) | 23 ± 1 | 75 ± 5 |
†
α-DsbA2ΔN: I(0) = 0.0427 ± 0.0001 cm−1, R g = 18.9 ± 0.1 Å, M = 23 kDa (2.5 mg ml−1); I(0) = 0.0890 ± 0.0002 cm−1, R g = 19.2 ± 0.1 Å, M = 24 kDa (5.0 mg ml−1). There is a small but significant systematic change in R g and M in the concentration range measured, but based on the trend a concentration of 1.25 mg ml−1 was deemed to be free of concentration-dependent effects. FL α-DsbA2: at the peak concentration of ∼2.2 mg ml−1, R g = ∼27.6 Å, which increases and plateaus at R g = ∼28.0 Å at a concentration of between 0.35 and 1.15 mg ml−1. This range was deemed to be free of concentration-dependent effects and all eight frames collected over this concentration range were combined, where the average concentration was 0.75 mg ml−1.