Applying 3D ED/MicroED workflows toward the next frontiers.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

Engineering of brick and staple components for ordered assembly of synthetic repeat proteins.

Synthetic ɑRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ɑRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.

Design, synthesis, and characterization of protein origami based on self-assembly of a brick and staple artificial protein pair.

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.

Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery.

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been 'domesticated', that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.