Single‐Particle Cryo‐EM and 3D Reconstruction of Hybrid Nanoparticles with Electron‐Dense Components

Abstract

Single-particle cryo-electron microscopy (cryo-EM), accompanied with 3D reconstruction, is a broadly applicable tool for the structural characterization of macromolecules and nanoparticles. Recently, the cryo-EM field has pushed the limits of this technique to higher resolutions and samples of smaller molecular mass, however, some samples still present hurdles to this technique. Hybrid particles with electron-dense components, which have been studied using single-particle cryo-EM yet with limited success in 3D reconstruction due to the interference caused by electron-dense elements, constitute one group of such challenging samples. To process such hybrid particles, a masking method is developed in this work to adaptively remove pixels arising from electron-dense portions in individual projection images while maintaining maximal biomass signals for subsequent 2D alignment, 3D reconstruction, and iterative refinements. As demonstrated by the success in 3D reconstruction of an octahedron DNA/gold hybrid particle, which has been previously published without a 3D reconstruction, the devised strategy that combines adaptive masking and standard single-particle 3D reconstruction approach has overcome the hurdle of electron-dense elements interference, and is generally applicable to cryo-EM structural characterization of most, if not all, hybrid nanomaterials with electron-dense components.

Cryo‐electron microscopy and cryo‐electron tomography of nanoparticles

“…3D cryo-TEM methods have been applied to a variety of DNA based nanoparticles including minicircles, 136 self-assembled DNA tetrahedrons, octahedrons, and symmetric polyhedra, [137][138][139] a nanoscale box, 140 various 3D DNA-origami objects, 141 nanocages, 142,143 and origami frames. 144 In a recent study, 3D cryo-TEM methods were used to evaluate the structure of octahedral DNA-origami frames with gold nanoparticles attached at all of the vertices or at specific vertices.…”

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

“…Specifically, 60 nm gold nanoparticles were found to be the most efficient for generating homogeneous Gag VLPs. However, while broad morphological features of the Gag shell assembled on gold nanoparticles appeared to mimic those of authentic immature HIV‐1, these VLPs were not amenable to detailed structural analysis using cryo‐EM since the electron‐dense gold core strongly absorbed and scattered electrons, obscuring any signal from the Gag lattice . Moreover, colloidal metal nanoparticles are not strictly monodisperse, exhibiting, on average, a 10% variation in size.…”

From binary AB to ternary ABC supraparticles

“…[45][46][47][48] Two-dimensional (2D) CryoTEM offers a direct and relatively easy way to visualize the structural characteristics of nanomaterials. While the structural characteristics of hybrid nanomaterials can be assessed with 2D CryoTEM images, a detailed and quantifiable determination of a three-dimensional (3D) structure requires cryo-ET, [49][50][51][52][53][54] which involves collecting a tiltseries of 2D projection images for a feature of interest, which can be utilized to generate a 3D tomogram. CryoET involves tilting the specimen through an angular range of typically À701 to +701 and collecting a tilt series of 2D projection images from a feature (in this context a hybrid nanoparticle) of interest.…”