To achieve our first objective, a new method was developed to purify the Sin3/Rpd3L HDAC complex from yeast S. cerevisiae. We benchmarked and optimized the tandem affinity purification (TAP) procedure to produce highly pure, fully-assembled, and high-yield samples suitable for biochemical/biophysical assays, mass spectrometry, and cryo-EM studies. The purified complexes exhibited the expected protein composition, minimal contaminations, and an average particle radius of about 14 nanometers. After stabilizing the complex by cross-linking, the sample was applied to microscope grids and Cryo-EM imaging was performed. We observed a good particle distribution on the grid and the collected data yielded a structure of the complex at a resolution of 3.37 Å. Importantly, results were independently repeated at least three times and structures at comparable resolutions (i.e. around 3.5 Å) were obtained in each case; showing the reproducibility of the established cryo-EM pipeline. To further leverage our findings, we are exploring the binding of the Sin3/Rpd3L HDAC complex to inhibitors and specific nucleosome substrates. Using our optimized protocol, we have already obtained a cryo-EM structure of the complex bound to an HDAC inhibitor (~3.5 Å), which revealed the binding mode in a multiprotein complex environment. Overall, these findings demonstrate the robustness of the protein purification/cryo-EM platform developed at the host institution during the MSCA project.
For the second objective, a human SIN3A HDAC complex (containing dozens of subunits) was purified using an established tandem affinity purification method in human cells. Cells were transfected with a plasmid co-expressing the tagged SUDS3 and SIN3A subunits, which were then captured using affinity beads. The purified complex contained expected components, with mass spectrometry analysis showing 80% purity, suitable for further studies. Samples and buffers were optimized to be compatible with cryo-EM studies and data collections on different purified fractions are expected to produce results in the near future.
