Computational protocol: Conformational sampling of membranes by Akt controls its activation and inactivation

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Protocol publication

[…] SAXS data were collected on Akt1 proteins using an in-line size exclusion chromatography setup on BM29 at the European Synchrotron Radiation Facility (ESRF). Proteins were applied to a Superdex 200 column equilibrated in 20 mM Tris, pH 7.4, 100 mM NaCl, and 1 mM Tris carboxyethyl phosphine (TCEP), and images were acquired every second for the duration of the size exclusion run. Buffer subtraction was performed by averaging 50 frames either side of the peak. All subsequent data processing steps were performed using the ATSAS data analysis software 2.8.2. The program DATGNOM () was used to generate the pair distribution function [P(r)] for each isoform and to determine Dmax and Rg from the scattering curves [I(q) vs. q] in an automatic, unbiased manner. Ab initio molecular envelopes for Akt1DrLink were computed by 10 iterative cycles of simulated annealing starting with a dummy atom model in DAMMIF (). The models were aligned, averaged, and filtered using DAMAVER (). The structure of Akt11−443 in complex with inhibitor VIII (PDB ID code 3O96) was compared with the scattering of Akt1DrLink using CRYSOL () and superimposed with the refined ab initio envelope using SUPCOMB (). For Akt1DA, rigid body modeling was performed using CORAL (), with PDB ID code 1UNP (PH domain) and PDB ID code 4EKK (chain A; kinase domain) as the starting rigid body models. Linker residues were implemented in CORAL as dummy residues. Iterative runs of CORAL were performed in which the kinase domain was allowed to move, while the PH domain was fixed. […]

Pipeline specifications

Software tools ATSAS, DAMMIF, CRYSOL
Application Small-angle scattering
Chemicals Sirolimus