Superose 12 hr10 30

The sample was prepared according to Borko et al. (2013 ▶ ). The sample quality was tested by dynamic light scattering (DLS; Zetasizer Nano-S) and size-exclusion chromatography (SEC; GE ÄKTA FPLC, Superose 12 HR10/30) at 5–10°C. Prior to SAXS data collection the protein sample was diluted at ratios of 1:1, 1:2, 1:3, 1:4 and 1:5 with the sample buffer without detergents. The protein concentration during the measurement was in the range 2–10 mg ml⁻¹. SAXS data were collected on beamline X33, DESY, Hamburg (see Supporting Information1 for beamline characteristics). The SAXS data were processed with programs from the ATSAS 2.5 package (Supplementary Fig. S1). Initial processing and analysis were performed with PRIMUS (Konarev et al., 2003 ▶ ). The radius of gyration (R_g) was estimated by Guinier approximation and from the pair-distribution function P(r). Information about the degree of protein disorder was obtained from the Kratky plot. The particle volume was calculated using the Porod invariant and the molecular weight was estimated from the Porod volume. Ab initio modelling was performed with GASBOR (Svergun et al., 2001 ▶ ) using ten repetitions with one scattering curve, with χ² between 0.9 and 1. The normalized spatial discrepancy (NSD) of all models was compared using DAMSEL (Volkov & Svergun, 2003 ▶ ). The average NSD was 1.50 ± 0.07.

Xenopus laevis adult females (Centre de Ressources Biologiques Xenopes, CNRS, France) were bred and maintained according to current French guidelines in the conventional IBPS aquatic animal facility, with authorization: Animal Facility Agreement: #A75-05-25. All experiments were subject to ethical review and approved by the French Ministry of Higher Education and Research (reference APAFIS#14127-2018031614373133v2).
All reagents, unless otherwise specified, were from Sigma. Okadaic acid (OA), magnetic GSH-beads and Co-beads were purchased from Enzo Life Sciences, Promega and Clontech Laboratories respectively. Uno Q-25 was purchased from Bio-Rad, Mono Q 4.6/100PE, Phenyl-Superose HR5/5 and Superose 12 HR10/30 were from GE Healthcare.

In all, 0.4 mg/ml of PAN was mixed with increasing amounts of Trypsin (0–2 μg per 40 μl rxn), incubated for 1 h at room temperature in reaction buffer (50 mM Tris pH 7.5, 5% v/v glycerol), and reactions were quenched with the manufacturer's recommendation amounts (1:100) of Halt Protease Inhibitor Cocktail (Thermo Scientific). Samples were checked for CC-OB domain fragment via Native-PAGE, and samples containing CC-OB domain fragment were pooled and injected onto a size exclusion column (Superose 12 HR 10/30, GE). Immediately after elution, 1:100 Halt Protease Inhibitor Cocktail (Thermo Scientific) was added to each fraction. Peaks were analyzed via SDS-PAGE and Native-PAGE and Fraction 1 (containing near-full-length PAN) and Fraction 3 (containing CC-OB fragment) were pooled. Fractions 1 and 3 were incubated with either 1 mM DTT (reduced sample) or 1 mM TT (oxidized sample) for 1 h at room temperature. After desalting (as described above), samples were run on SDS-PAGE and visualized via silver stain (Pierce Silver Stain Kit, Thermo Scientific). Representative images from four independent experiments are shown.