Chapter 7: Structure Determination and Refinement of Large Macromolecular Assemblies at Low Resolution

The COP9 signalosome (CSN) is a large multi-protein complex in eukaryotes that is an essential regulator of intracellular protein degradation. Structures of human CSN were determined by X-ray crystallography from weakly diffracting crystals variably affected by twinning and rotational pseudo-symmetry. Strategies were developed to overcome further challenges of crystal instability and non-isomorphism that were strongly influenced by handling, radiation damage, and difficulty obtaining heavy atom derivatives. Many subunits of the same fold class were distinguished at low-resolution aided by combinatorial selenomethionine labelling. Here, a detailed account of the crystallographic structure determination of CSN is given as it was approached using low-resolution cluster compound MIRAS phasing, MR-SAD phasing with electron density models, and cross-crystal averaging and co-refinement of multiple structures, exploiting non-isomorphism across unit-cell variants of the same crystal form.