Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

Ramasubramanian Sundaramoorthy; Amanda L. Hughes; Hassane El-Mkami; David Norman; Tom Owen-Hughes

The appropriate ratio of ScChd1(1-1305∆57-88) to nucleosome for 1:1 and the 2:1 complex formation in the presence of 5-fold molar excess of ADP-BeFx was determined by titration and native PAGE analysis. The formed complex was then purified by size exclusion gel filtration using a PC 3.2/30 superdex 200 analytical column in 20mM Tris, 50mM NaCl and 250 µM ADP.BeFx . In a typical run 50uLs of 20 µM of sample was injected using Dionex autoloader. 50uLs fractions were collected and analysed in 6% Native PAGE gel and appropriate fractions containing ScChd1-nucleosome complexes were pooled together. A 4 µl drop of sample was then applied to C-flat Holey carbon foil (400 mesh R1.2/1.3 uM) pre-cleaned with glow discharge (Quorum technologies). After 15 second incubation, grids were double side blotted for 4 s in a FEI cryo-plunger (FEI Mark III) at 90% humidity and plunge frozen into −172 °C liquefied ethane. Standard vitrobot filter paper Ø 55/20 mm, Grade 595 was used for blotting.