Computational protocol: Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid–virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

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Protocol publication

[…] Sequential backbone signal assignment was first carried out by combined analyses of the 3D triple-resonance spectra HNCA (–) and HN(CO)CA (,), and by evaluation of sequential nuclear Overhauser effects (NOEs) in a 15N-edited NOESY spectrum (–), acquired with a mixing time of 100 ms. Complete HN, N, Cα and Hα resonance assignments of all 15N-HSQC-detected residues were subsequently achieved based on 15N-edited TOCSY spectrum (), acquired with a mixing time of 50 ms. Four glutamine and one asparagine side chain amides were detected and unambiguously assigned in the 15N-edited NOESY spectrum. The indolic HN group of the only Trp residue was also unambiguously assigned. All NMR data were processed with the software VNMRJ 1.1.D (Varian Inc.). 2D and 3D spectra were analyzed using tools available in CARA software ().Secondary structural elements of Stf76 were initially identified by chemical shift index (CSI) analysis (), which was generated using Cα and Hα chemical shifts. Dihedral angle restraints were calculated from HN, Cα, Hα and N chemical shifts with the software TALOS+ ().The structure calculation was performed with the program CS-Rosetta (,) using as structural restraints the torsion angles ϕ/ψ derived from TALOS+ database and the HN, Cα, Hα and N chemical shifts of those residues indicated by TALOS+ to be rigid in pico-second timescale with an order parameter S2 > 0.7. A set of 200 fragment candidates matching these chemical shifts was used to calculate 3000 structures in Rosetta. The energy of these Rosetta structures was then rescored against the observed chemical shifts and the 20 conformers with the lowest rescored energy were selected for the ensemble. The structures were visualized and evaluated by using the programs MOLMOL (), PROCHECK-NMR () and MOLPROBITY ().The most representative model of the Stf76 structure was compared to structures in the Protein Data Bank by using the DaliLite server ().15N relaxation parameters of Stf76 were evaluated by recording inversion recovery 1H-15N HSQC for R1 measurements and spin echo 1H-15N HSQC for R2 measurements. Acquisition parameters and relaxation data processing and analysis were essentially as reported (,). An estimate of the isotropic correlation time, τc, of Stf76 was obtained from the ratios of 15N R2 and R1 values of residues that passed the coarse and fine-filtering steps (), using the equation reported (). The Stokes–Einstein–Debye equation was then used to calculate the hydrodynamic radius from the τc. The hydrodynamic properties were also evaluated using the NMR software hydropro (). […]

Pipeline specifications

Software tools TALOS+, CS-Rosetta, MOLMOL, PROCHECK, MolProbity
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Sulfolobus islandicus