Gold nanoparticle–protein arrays improve resolution for cryo-electron microscopy

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.

RETRACTED: High-Affinity Gold Nanoparticle Pin to Label and Localize Histidine-Tagged Protein in Macromolecular Assemblies

“…However, these approaches not only suffer from a lack of specificity due to multiple lysine and cysteine residues within macromolecular assemblies; but also compared to non-covalent labeling, these chemical reactions are 2–3 orders of magnitude slower, thus requiring higher concentrations and longer incubation times, which increase the probability of non-specific modifications. In an attempt to combine the advantages of a small chemical recognition unit with fast and specific non-covalent interaction, AuNPs conjugated to mono -nitrilotriacetic acid ( mono -NTA) (Figure 1A) have been employed to bind proteins via a oligohistidine-tag (His-tag) (Hu et al, 2007, Hu et al, 2008). Compared to chemical coupling, capturing via Ni-NTA/His-interaction is much faster (~1×10 5 M −1 s −1 ), i.e.…”

Functionalized Gold Nanoparticles and Their Biomedical Applications

“…Besides the usual spherical shape, GNPs have been synthesized in various other shapes affecting their physical and biochemical properties. For example, hexagon and boot shaped GNPs show different surface enhanced Raman scattering (SERs) which in turn can be used to detect molecules conjugated to GNPs such as avidin, thereby making these functionalized GNPs (fGNPs) useful for biolabelling, bioassay, clinical diagnosis and therapy [ 17 ]. Gold nanocages of six and eight facets have also been synthesized [ 18 ].…”

Biomolecular Imaging at High Spatial and Temporal Resolution In Vitro and In Vivo

“…This is because the defocus can be chosen so that the Thon rings coincide with the location of the diffraction maxima. Indeed, forcing single particles into 2D arrays has shown promise in increasing the resolution of averaged 2D images of proteins with a decreased number of particles [73].…”