Computational protocol: Structural analysis of the complex between influenza B nucleoprotein and human importin α

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Protocol publication

[…] All datasets were collected on BM29 (ESRF). For in-line SEC-SAXS, the experimental setup consists of High Pressure Liquid Chromatography (HPLC) system connected to an analytical SuperdexTM increase 200 5/150 GL column (GE Healthcare) followed down-stream by SAXS sample capillary. SAXS measurements were performed every second with a Pilatus 1 M detector (distance of 2.87 m) allowing a q range of 0.03 to 4.5 nm with a wavelength of 0.01 nm.Experimental curves were subtracted and analyzed using Primus (ATSAS programs suite). To verify the molecular mass, the Rambo and Tainer method was used,. Rg predictions using Guinier extrapolation were plotted against the elution volume to select the most monodisperse part of the protein elution peak. SAXS datasets within this zone were scaled and averaged to produce one unique I(q) curve. Distance distribution functions p(r) were calculated using the program GNOM. The ab-initio models were generated by MONSA using when available, the individual data sets in order to fit them simultaneously. Homologue PDB structure comparison was assessed using Crysol. Homologue structure fitting within the DAMAVER envelope was performed with PyMOL and curve representations using Graphpad (Prism). SAXS curves with B/NP alone contained the poly-His tag, which is cleaved off for all samples probing interaction with importin-α.A description of the conformational ensemble of full length B/NP was obtained using the crystal structure of the folded domain (PDB 3TJ0) and Flexible-Meccano to add the intrinsically disordered tail as a conformational ensemble. The backbone dihedral angle distribution identified by a Flexible-Meccano/ASTEROIDS combination to describe B/NPTAIL (see above) was assumed to be valid also within the full length protein construct. 200 conformers were generated and SAXS curves for each of the conformers were calculated using CRYSOL and averaged to obtain the expected SAXS curve for the full length B/NP ensemble. […]

Pipeline specifications

Software tools MONSA, CRYSOL, ATSAS, PyMOL
Application Small-angle scattering