Heavy atom labels can augment electron microscopy of biological structures by: a) revealing the site on a specific structure within a complex, and b) facilitating location and alignment of an overall complex. A useful EM label has two components: 1) a dense object which gives a high signal-to-noise ratio under the circumstances of observation, and 2) a linker which confers specificity, rigidity, and high occupancy without affecting biological function. Previously we have optimized labels for STEM. In this proposal we will better adapt the technology to cryoEM and modern molecular biology. New technology developed for protein engineering provides a variety of tags which can be inserted into the structure genetically and used for purification. These include: His-tags, Flag-tags, GST-tags, and strept-tags. Some of these same tags appear ideally suited as linkers for rigid, high-affinity attachment of density labels. Therefore, we propose to develop a labeling toolkit for cryoEM based on these new capabilities. In particular we will primarily evaluate five tags that bind to specialized gold clusters: 1) Ni-NTA-gold binding to 6x-His tags, 2) negatively charged gold to 6x Arg tags, 3) monomaleimido gold linked to cysteine, 4) arsenic-gold to tetra-Cys tags, and 5) gold targeted to a gold-binding sequence. These labels will then be used to determine subunit positions in several important protein complexes: The DNA repair complexes consisting of subcomplexes: human Ku70/Ku80/DNA-PK complex, human ligase IV/Xrcc4 complex, human RAD50/Mre11/Nbs1 complex, and the human chromatin remodeling complex ACF. The gold labels will additionally be used to better align the complexes to improve reconstructions from conical tilt pairs using cryoEM.
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