Cryo-electron microscopy of cloneable inorganic nanoparticles

Abstract

Our understanding of biology is best understood through direct, empirical measurements of biomacromolecules and biological systems. The functions of proteins are directly linked to both their structure and their intracellular organizations. Single particle cryo-electron microscopy has revolutionized modern structural biology by enabling the structural determination of proteins and protein-complexes in purified samples without the need to form large crystals as required by X-ray crystallography. With single particle cryo-EM, atomic and near-atomic resolution structures are now routine which offer insight into the functions of biomacromolecules. While these insights are invaluable, there is increasing momentum for integrative structural biology which aims to accomplish structural determination of biomacromolecules in their cellular, tissue, or organismal context. There remains a grand challenge in biological imaging where biological materials have low innate contrast. Cloneable contrast labels that impart contrast to discrete protein densities do not reliably exist for cryo-electron microscopy. In contrast, fluorescent proteins are reliable and routine for localizing fluorescent protein / protein of interest genetic fusions in visible-light microscopies. We have proposed and developed intracellularly synthesized inorganic nanoparticles called 'cloneable nanoparticles' as a solution to this grand challenge. Cloneable nanoparticles are inorganic nanoparticles, synthesized by a protein/peptide (or combination thereof) which controls and defines the properties of the inorganic nanoparticle. Here we have defined the cloneable nanoparticle paradigm and described the development of a cloneable selenium nanoparticle. Further, we show the application of the cloneable selenium nanoparticle as a cloneable contrast label for biological electron microscopy and correlative light-electron microscopy and detail progress towards adapting the cloneable selenium nanoparticle for use in cryo-electron tomography. With the aim to later expand cloneable nanoparticles to include a myriad of orthogonal cloneable contrast labels (analogous to different colored fluorophores), and to gain understanding about enzymatic nanoparticle synthesis, a single particle cryo-EM study is on-going. Lastly, we have shown the application of directed evolution for cloneable nanoparticles, suggesting that this is a viable path, alongside rational protein design, towards developing future cloneable nanoparticle cryo-electron microscopy labels.