How a mutation in the SKD3 enzyme can cause MGCA7 disease BCMHouston NatureComms
have excellent stereochemical properties with 100.0% and 98.9% of residues in the Ramachandran favored region.The ATPase activity of mature SKD3 and variants was determined at 25 °C in 25 mM HEPES-KOH pH 8.0, 150 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT, and 2 mM ATP using the malachite green colorimetric assay.
For the fiber disaggregation assay, α-syn fibrils were thawed and spun down three times at 20,000 x g for 20 min at 4 °C. To ensure that no soluble α-synuclein-GFP was present due to spontaneous resolubilization, the supernatant was removed after each cycle and fibrils were resuspended in refolding buffer. For the assay, α-syn fibrils were mixed with 5 μM SKD3 and incubated with an ATP regenerating system in refolding buffer at 30 °C for 90 min.
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