Table 1 Overall SAXS data
From: Structural insights into the substrate binding mechanism of the class I dehydratase MadB
SAXS device | P12, PETRA III, DESY Hamburg1 | |
|---|---|---|
Data collection parameters | ||
Detector | PILATUS 6 M | PILATUS 2 M |
Detector distance (m) | 3.0 | 2.827 |
Beam size (µm × µm) | 120 × 200 | 200 × 200 |
Wavelength (nm) | 0.124 | 0.099 |
Sample environment | Quartz capillary, 1 mm ø | Quartz capillary,1 mm ø |
s range (nm−1)‡ | 0.02–7.0 | 0.025–6.0 |
Absolute scaling method | Comparison with scattering from pure H2O | |
Normalization | To transmit intensity by the beam-stop counter | |
Scattering intensity scale | Absolute scale, cm−1 | |
Sample | apo MadB | MadB with MadL3 |
Organism | Clostridium sp. Maddingley | |
UniProt ID | K6TUQ9 (MadB), K6SWQ2 (MadA) | |
Mode of measurement | Online SEC-SAXS | |
Temperature (°C) | 15.0 | 10.0 |
Protein buffer | 50 mM HEPES pH 8.0, 200 mM NaCl, 1% (v/v) Glycerol | |
SEC-Column | Superdex 200 Increase 10/300 GL | Superdex 200 Increase 3.2/300 GL |
Injection volume (µl) | 100 | 50 |
Flowrate (ml/min) | 0.5 | 0.075 |
Exposure time (# frames) | 0.995 s (3000) | 2 s (1200) |
# frames used for averaging | 55 | 16 |
Protein concentrations (mg/ml) | 6.00 | 10.00 |
Substrate | - | 400 µM Leader 3 |
Structural parameters | ||
Guinier Analysis (PRIMUS) | ||
I(0) ± σ (cm−1) | 0.12 ± 0.000066 | 262.51 ± 0.192 |
Rg ± σ (nm) | 4.48 ± 0.005 | 4.47 ± 0.005 |
s-range (nm−1) | 0.091–0.246 | 0.087–0.288 |
min < sRg < max limit | 0.41–1.10 | 0.39–1.28 |
Data point range | 6–62 | 8–47 |
Linear fit assessment (R2) | 0.999 | 0.999 |
PDDF/P(r) Analysis (GNOM) | ||
I(0) ± σ (cm−1) | 0.12 ± 0.000076 | 260.80 ± 0.165 |
Rg ± σ (nm) | 4.30 ± 0.003 | 4.41 ± 0.003 |
Dmax (nm) | 13.57 | 13.78 |
Porod volume (nm3) | 439.58 | 445.04 |
s-range (nm−1) | 0.188–5.335 | 0.108–4.670 |
χ2 / CorMap P-value | 1.04/0.106 | 1.09/0.194 |
Molecular mass (kDa) | ||
From I(0) | n.d. | n.d. |
From Qp4 | 272.25 | 283.46 |
From MoW25 | 212.43 | 264.39 |
From Vc6 | 250.32 | 261.37 |
Bayesian inference7 | 242.63 | 318.45 |
From sequence | 249.72 (dimer) | 253.17 (dimer + substrate) |
Shape modeling/software | ||
Gasbor | ||
s-range (nm−1) | 0.188–5.332 | 0.108–4.665 |
χ2/CorMap P-value | 1.42/0.0000000001 | 1.27/0.00000005 |
SASBDB accession codes8 | SASDRX6 | SASDRY6 |
Software | ||
3.0.4 | ||
Primary data reduction | ||
Data processing | GNOM13 | |
Ab initio modeling | GASBOR14 | |
Superimposing | SUPCOMB15 | |
Model visualization | PyMOL16 | |
