Computational protocol: Ligand binding and aggregation of pathogenic SOD1

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Protocol publication

[…] Crystals grown in the P21 space group were soaked with compounds (50 mM) dissolved in 2.5 M (NH4)2SO4, 150 mM NaCl, 100 mM NaOAc pH 4.75 solution for 2 h at 20 °C. Crystals grown at pH 8 in the C2221 space group were soaked in compounds (50 mM) dissolved in 2.5 M (NH4)2SO4, 150 mM NaCl, 100 mM Tris-HCl pH 8. X-ray crystallographic data sets were collected at the Diamond Light Source stations I02 an I03, Oxford, UK, and at the Soleil station Proxima 1, Saint-Aubin, France. Reflection data were integrated and scaled using HKL2000 (ref. ), XDS, or iMosflm and Scala. Cycles of model building and refinement proceeded using REFMAC5 and COOT. Crystallographic data collection, scaling and refinement statistics for I113T SOD1 in the P21 space group are presented in . [...] Monomeric copper-apo, zinc-holo A4V SOD1 was determined to be homogenous by SDS–PAGE and its was identity confirmed by peptide mass fingerprinting. It was analysed using the combined HPLC–SAXS setup at beamline SWING as follows: 100 μl of 350 μM protein was loaded onto an Agilent Bio SEC-5 HPLC column, using an Agilent 1200 HPLC at 200 μl min−1 at 10 °C. Small-angle scattering parameters were calculated for a series of 250 exposures (starting at 3 ml and lasting 1,000 ms separated by 500 ms dead time) taken as the protein eluted with 100 frames taken before the column void volume for buffer subtraction. The incident beam had wavelength 1.03 Å and the sample-detector distance was 1.782 m. One hundred buffer frames were averaged and subtracted from ten frames corresponding to an elution volume of 3.68–3.73 ml, which were then averaged to yield the scattering profile. A Guinier approximation was performed with Primus, and the distance distribution function was calculated using q range up to 0.4 Å−1 with GNOM. Twenty ab initio models were constructed using GASBOR and were averaged with DAMAVER. […]

Pipeline specifications

Software tools XDS, iMosflm, CCP4, REFMAC5, Coot, ATSAS, GASBOR
Applications Small-angle scattering, Protein structure analysis
Diseases Amyotrophic Lateral Sclerosis
Chemicals Copper, Isoproterenol, Superoxides, Zinc