Computational protocol: The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil

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Protocol publication

[…] SAXS measurements were carried out at the BM-29 BioSAXS beamline at the ESRF Storage Ring (Grenoble, France) using a sample-to-detector distance of 2.87 m []. KISS1 samples were measured at 10°C (to minimize radiation damage) and concentrations of 4.0 and 1.0 g/L in PBS, pH 7.0, 1 mM DTT. The scattering pattern of the buffer solution was recorded before and after measuring each protein sample. The obtained scattering profiles covered a momentum transfer range of 0.003 < s < 0.49 Å-1. A comparison of ten consecutive X-ray exposures indicated the absence of radiation damage. The final curves at each concentration were derived after subtracting the averaged buffer from the protein patterns using standard protocols within PRIMUS []. SAXS curves at both concentrations were merged to derive a final profile that was used for the subsequent structural analysis. The forward scattering, I(0), and the radius of gyration, Rg, were evaluated using Guinier's approximation [], assuming that at very small angles (s < 1.3/Rg), the intensity can be described as I(s) = I(0) exp(−(sRg)2/3). The pairwise distance distribution function, p(r), and the maximum particle diameter, Dmax, were computed with GNOM [] using a momentum transfer range of 0.013 < s < 0.29 Å-1.The ensemble optimization method (EOM) was used to describe the overall properties of KISS1 in solution []. To this aim a pool of 10,000 conformations was built with Flexible-Meccano [, ] and side-chains were added with Sccomp []. For each conformation the theoretical SAXS curve was computed with Crysol [] and used for the EOM sub-ensemble optimization. Two hundred independent EOM runs with 50 selected conformers were performed using the χ2 statistical test as the selection criterion. The resulting ensemble composed of 10,000 conformations was structurally evaluated using its Rg distribution. […]

Pipeline specifications

Software tools ATSAS, EOM, CRYSOL
Application Small-angle scattering
Diseases Neoplasms