Computational protocol: Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation

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[…] SAXS experiments were conducted using a Rigaku BioSAXS-2000 home source system with a Pilatus 100K detector and a HF007 copper rotating anode (Rigaku Americas, The Woodlands, TX). Data were collected at a fixed sample-to-detector distance using a silver behenate calibration standard. The instrument software was used to reduce the data to scattering intensity, I(Q), vs. wave vector transfer, Q ( = 4π sin(θ)/λ, where 2θ is the scattering angle), and then subtract the buffer background. The scattering curves were collected at a protein concentration of 30 µM, at 25 °C, for 1 h. The reduced data were analyzed with the ATSAS suite, using PRIMUS and GNOM for Guinier and P(r) analyses, with a q range between 0.018 and 0.31 Å−1. Reported Rg values were obtained from the P(r) analysis performed with GNOM, although similar values were obtained using the Guinier approximation performed with Primus. [...] The SASSIE program was used to generate ensemble models for NPM1N188 to compare with the SAXS data. Using the pentameric core structure in PDB ID 4N8M as the starting model, the remaining NPM1N188 sequence was generated for each subunit using the psfgen package within VMD/NAMD. SASSIE then was used to minimize the structure and run Monte Carlo simulations with the pentamer core kept fixed (PDB ID: 4N8M). The models were then compared to the SAXS I(Q) curve for NPM1N188 (150 mM NaCl) to obtain χ2 values. We sampled the potential range of the conformational landscape with SASSIE to create representative conformers and then compared each conformer to the experimental curve to obtain a χ2 value. The χ2 vs. Rg plot (Supplementary Fig. ) exhibits a broad basin near the experimentally observed Rg value of 44 Å, suggesting that NPM1N188 dynamically exchanges between conformers with a range of compaction values. However, because the SAXS curves reflect only the ensemble averaged conformation, we cannot define minimum and maximum compaction limits for NPM1N188 molecules that experience conformational averaging. […]

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