Assessment of protein-protein interfaces in cryo-EM derived assemblies

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we comprehensively assessed protein–protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed Protein Interface-score (PI-score), a density-independent machine learning-based metric, trained using the features of protein–protein interfaces in crystal structures. We evaluated 5873 interfaces in 1053 PDB-deposited cryo-EM models (including SARS-CoV-2 complexes), as well as the models submitted to CASP13 cryo-EM targets and the EM model challenge. We further inspected the interfaces associated with low-scores and found that some of those, especially in intermediate-to-low resolution (worse than 4 Å) structures, were not captured by density-based assessment scores. A combined score incorporating PI-score and fit-to-density score showed discriminatory power, allowing our method to provide a powerful complementary assessment tool for the ever-increasing number of complexes solved by cryo-EM

A survey of the use of iterative reconstruction algorithms in electron microscopy

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET)

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized gt 20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing gt 700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3212