A statistical coil ensemble of P1–304 comprising 10,000 structures was generated using flexible-meccano. Two hundred conformations that best described the experimentally obtained N, HN, Cα, Cβ, and CO chemical shifts were selected from the ensemble using the genetic algorithm ASTEROIDS. A new ensemble of 8500 conformers was generated on the basis of the Φ and Ψ angles of the selected conformers, supplemented with 1500 conformers from the initial statistical coil ensemble (28, 29). This new ensemble was subjected to another round of ASTEROIDS selection, and the iteration step was repeated eight times until the ensemble converged with respect to the chemical shifts. Ensemble averaged chemical shifts were calculated using SPARTA (54), and SAXS curves were obtained using CRYSOL (55). Another round of ASTEROIDS was used to include, in addition to chemical shifts, the SAXS curves into the selection.

To build a conformational model of P1–304N1–525 and N1–405P1–304, the structure of NCORE and the first 50 residues of P in the complex were adapted from the x-ray structure of MeV (14) and NiV (15) N0P. The N-terminal flexible arm of N, NTAIL, and PNTD were subsequently built onto the structure using flexible-meccano with the helical sampling of the NTAIL molecular recognition element, as previously described (56), as well as the Φ and Ψ angles of the last selection of P1–304 to describe PNTD within the complex. The order by which the different chains were built onto the folded structure did not make a difference for the final ensemble of 100,000 conformers. ASTEROIDS selections with sizes of 5 to 50 conformers were then selected on the basis of the SAXS scattering curve. The same procedure was applied to describe the scattering curve of P1–304,HELL→AAAAN1–525 and P1–304,HELL→AAAAN1–405. To illustrate the interaction between the HELL motif within P1–304 and N, 200 conformers were filtered that fulfill a distance constraint of 5 Å between the P1–304 HELL motif and its binding site on N (Fig. 5F).

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