STAT2 protein, human

Guided by the cryo‐EM density map 1 (Appendix Fig S3) and XL‐MS distance restraints (Fig 2C), a partial molecular model of E27 was generated de novo using the program Coot 0.9.5 (Emsley & Cowtan, 2004 (link); Emsley et al, 2010 (link)). In addition, in silico AlphaFold2 structure prediction of E27 was performed (Jumper et al, 2021 (link)) using the ColabFold web server (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb; Mirdita et al, 2022 (link)). The AlphaFold2 output coordinates were in very good agreement with the experimentally determined E27 structure (RMSD of 1.489 Å, 2764 aligned atoms). Accordingly, the model was completed by merging the initial model with the AlphaFold2 template and adjusting backbone and side chain positions where necessary using Coot 0.9.5. DDB1 coordinates were taken from a previously solved DDB1/DCAF1‐CtD complex structure (Banchenko et al, 2021 (link)), rigid body‐fitted and adjusted manually using Coot 0.9.5. The coordinates of the rnSTAT2 CCD were extracted from the AlphaFold protein structure database (https://alphafold.ebi.ac.uk/entry/Q5XI26; Varadi et al, 2022 (link)), rigid body‐fitted and adjusted manually using Coot 0.9.5. As final step, automated real space refinement was performed using the program Phenix 1.19.2–4158 (Liebschner et al, 2019 (link)). The refined DDB1/E27/STAT2 CCD structure was then fitted as rigid body in cryo‐EM density map 2 (Appendix Fig S3). Full‐length rnSTAT2 coordinates (https://alphafold.ebi.ac.uk/entry/Q5XI26) were placed by structural superposition of the CCDs. The STAT2 NTD and TAD were removed, and flexible loops in the SH2 domain were trimmed, because they could not be assigned to the experimental density, indicating that they are flexibly attached and mobile relative to the other STAT2 domains. Lastly, a chain break was introduced between STAT2 residues 315 and 317, and the STAT2 portion encompassing residues 317–701 (DBD, LD and SH2) was once more separately fitted as rigid body, to account for a small movement relative to the CCD. Subsequently, the model was subjected to rigid body and grouped b‐factor real space refinement using Phenix 1.19.2–4158, followed by molecular dynamics flexible fitting using the Namdinator server (Kidmose et al, 2019 ), applying default parameters.

A sample containing 7 μM HisSUMO‐E27, 7 μM GST‐DDB1_ΔBPB and 7 μM rnSTAT2 was incubated with 0.5 mg 3C protease in a volume of 1 ml buffer containing 10 mM HEPES‐NaOH pH 7.8, 150 mM NaCl, 4 mM MgCl₂, 0.5 mM TCEP, for 12 h on ice. Subsequently, the sample was subjected to GF chromatography on an Äkta prime plus FPLC (Cytiva), using a Superdex 200 16/600 column (Cytiva), connected in line to a 1 ml Glutathione Sepharose^® 4 Fast Flow column (Cytiva), at a flow rate of 1 ml/min, using the same buffer as eluent. GF fractions were analysed by SDS–PAGE. Fractions corresponding to the elution peak containing E27, DDB1_ΔBPB and rnSTAT2 were pooled and concentrated to 70 μl (protein concentration 4.1 mg/ml).
Suberic acid bis(N‐hydroxysuccinimide ester; DSS) cross‐linker (Merck) was added 1:1 (w/w) from a 100 μg/ml stock solution in DMSO, and the sample volume was adjusted to 400 μl with buffer containing 10 mM HEPES‐NaOH pH 7.8, 150 mM NaCl, 4 mM MgCl₂ and 0.5 mM TCEP. The sample was incubated for 1 h at 22°C, and the cross‐linking reaction was quenched by the addition of 20 μl 1 M Tris–HCl pH 7.8 for 10 min at 22°C. Precipitate was removed by centrifugation at 16,000 g, 22°C for 5 min. The supernatant was loaded on a Superdex 200 Increase 10/300 GL GF column, equilibrated in 10 mM Tris–HCl pH 7.8, 150 mM NaCl, 4 mM MgCl₂ and 0.5 mM TCEP, at a flow rate of 0.5 ml/min, using an Äkta pure FPLC (Cytiva). 0.5 ml fractions were collected and analysed by SDS–PAGE. Fractions containing the cross‐linked DDB1_ΔBPB/E27/STAT2 complex were pooled, concentrated to 1.3 mg/ml, flash‐frozen in 5 μl aliquots in liquid nitrogen and stored at −80°C.
R1.2/1.3400 mesh Cu holey carbon grids (Quantifoil) were glow‐discharged for 30 s using a Harrick plasma cleaner with technical air at 0.3 mbar and 7 W. 3.5 μl solution containing 0.5 μM (0.13 mg/ml) cross‐linked DDB1_ΔBPB/E27/STAT2 complex were applied to the grid, incubated for 45 s, blotted with a Vitrobot Mark IV device (Thermo Fisher) for 1–2 s at 4°C and 99% humidity and plunged in liquid ethane. Grids were stored in liquid nitrogen until imaging.