Computational protocol: Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

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Protocol publication

[…] SAXS experiments were performed on the SWING beamline at synchrotron SOLEIL (Saint-Aubin, France). The beam wavelength was λ = 1.033 Å. The 17 × 17 cm2 low-noise Aviex CCD detector was positioned at a distance of 2107 mm from the sample with the direct beam off-centered. The resulting exploitable q-range was 0.005–0.35 Å−1, where the wave vector q = 4π sin θ/λ and 2θ is the scattering angle.WT mt-AspRS and mutants Q184K and R263Q at, respectively, 7.4, 10 and 14 mg/ml in 50 mM HEPES-Na pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 0.1 mM EDTA and 1 mM DTT, were separated by SEC and analyzed by SAXS online. Thus, 45 μl protein solution was loaded onto an Agilent Bio SEC-3 column (300 Å, 4.6 × 300 mm, 3 μm) installed on an Agilent HPLC system and maintained at 15 °C. Proteins were eluted at a flow rate of 0.2 ml/min with a mobile phase containing 100 mM HEPES-Na pH 7.5, 250 mM NaCl, 5% (v/v) glycerol and 1 mM TCEP. The eluate was analyzed by SAXS in a continuous flow capillary cell with a frame duration of 1000 ms at intervals of 500 ms. Data processing, analysis and modeling step were done with PRIMUS and other programs of the ATSAS suite.Radii of gyration (Rg) were derived from Guinier approximation and used to estimate the molecular Porod volume. Rg was also calculated from the entire scattering pattern using the indirect transform package GNOM, which provides the distance distribution function p(r) of the particle. A dimeric mt-AspRS model including all residues (two polypeptide chains encompassing residues 41–645 plus a linker of 5 residues and a 6-His-tag) were derived from the X-ray structure (PDBid: 4AH6). The C-terminal tails added to the monomers consisted of the last 15 residues not visible in the crystal structure and of the 11 amino acid affinity tag. The conformational space of these tails was modeled under SAXS constraints using DADIMODO, a genetic algorithm-based refinement analysis program. The core of the dimer corresponding to the crystal structure was treated as a rigid body while the conformation of the C-terminal tails was explored by simulated annealing. A set of eight models were selected that best fit the experimental data. The goodness-of-fit was estimated using CRYSOL. […]

Pipeline specifications

Software tools ATSAS, CRYSOL
Application Small-angle scattering
Organisms Homo sapiens
Diseases Neurodegenerative Diseases, Leukoencephalopathies
Chemicals Amino Acids