Protein Crystallography: Challenges and Practical Solutions
Chapter 7: Structure Determination and Refinement of Large Macromolecular Assemblies at Low Resolution
Published:19 Jun 2018
Special Collection: 2018 ebook collectionSeries: Chemical Biology
R. D. Bunker, in Protein Crystallography: Challenges and Practical Solutions, ed. K. Beis and G. Evans, The Royal Society of Chemistry, 2018, ch. 7, pp. 157-180.
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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.