Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Yeast Cells

We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in yeast cells expressing metallothionein (MTn) tags (Figure 1). This approach should be useful for EM visualization of single-molecule in yeast cells, and easier adapted for bacterial cells.