Structure determination of conformationally variable proteins can prove challenging even when many possible molecular‐replacement (MR) search models of high sequence similarity are available. Calmodulin (CaM) is perhaps the best‐studied archetype of these flexible proteins: while there are currently ～450 structures of significant sequence similarity available in the Protein Data Bank (PDB), novel conformations of CaM and complexes thereof continue to be reported. Here, the details of the solution of a novel peptide–CaM complex structure by MR are presented, in which only one MR solution of marginal quality was found despite the use of 120 different search models, an exclusivity enhanced by the presence of a high degree of hemihedral twinning (overall refined twin fraction = 0.43). Ambiguities in the initial MR electron‐density maps were overcome by using MR‐SAD: phases from the MR partial model were used to identify weak anomalous scatterers (calcium, sulfur and chloride), which were in turn used to improve the phases, automatically rebuild the structure and resolve sequence ambiguities. Retrospective analysis of consecutive wedges of the original data sets showed twin fractions ranging from 0.32 to 0.55, suggesting that the data sets were variably twinned. Despite these idiosyncrasies and obstacles, the data themselves and the final model were of high quality and indeed showed a novel, nearly right‐angled conformation of the bound peptide.
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