Effect of Local Heterogeneities on Single-Layer DNA-Directed Protein Lattices Through Non-Averaged Single-Molecule 3D Structure Determination

Programmable and self-assembled two-dimensional (2D) protein lattices hold significant potential in synthetic biology, nanoscale catalysis, and biological devices. However, achieving high-order 2D lattices from three-dimensional (3D) nanoscale objects remains challenging due to structural heterogeneity caused by the flexibility and distortions of building blocks and their connectivity in a unit cell, leading to the formation of lattices with imperfections. This flexibility largely limits the analysis of key structural parameters at unit-cell resolutions due to the need to average 3D reconstructions in current methods. Here, we utilized advances in individual-particle cryo-electron tomography (IPET) to analyze the 3D structure of a designed 2D lattice formed by DNA-origami octahedral cages (unit-cell particles) encapsulating ferritin by determining the non-averaged 3D structure of each unit-cell particle. These protein-carrying DNA cages were analyzed at ferritin loading percentages of 100%, 70%, and 0%. Correlation analysis revealed that neither the ferritin loading percentage nor off-centralized placement in cages significantly affected lattice parameters, flexibility, or long-range order. Instead, the soft nature of DNA cages and interparticle linkages were the primary reasons for lattice imperfections. Structural improvements for enhancing lattice orders were evaluated through a series of molecular dynamics simulations. The developed cryo-EM 3D imaging reveals the molecular origin of heterogeneity of DNA-origami 2D lattices and highlights a path toward improved lattice designs.