Abstract
Cryo-electron microscopy single particle analysis shows limited resolution due to poor alignment precision of noisy images taken under low electron exposure. Certain advantages can be obtained by assembling proteins into two-dimensional (2D) arrays since protein particles are locked into repetitive orientation, thus improving alignment precision. We present a labeling method to prepare protein 2D arrays using gold nanoparticles (NPs) interconnecting genetic tag sites on proteins. As an example, mycobacterium tuberculosis 20S proteasomes tagged with 6x-histidine were assembled into 2D arrays using 3.9-nm Au NPs functionalized with nickel-nitrilotriacetic acid. The averaged top-view images from the array particles showed higher resolution (by 6-8A) compared to analysis of single particles. The correct 7-fold symmetry was also evident by using array particles whereas it was not clear by analysis of a comparable number of single particles. The applicability of this labeling method for three-dimensional reconstruction of biological macromolecules is discussed.