TomoNet: A streamlined cryoET software pipeline with automatic particle picking on flexible lattices.

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and 3D classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in one-, two- or three-dimensional arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other employing deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice.

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

Hierarchical organization and assembly of the archaeal cell sheath from an amyloid-like protein.

Certain archaeal cells possess external proteinaceous sheath, whose structure and organization are both unknown. By cellular cryogenic electron tomography (cryoET), here we have determined sheath organization of the prototypical archaeon, Methanospirillum hungatei. Fitting of Alphafold-predicted model of the sheath protein (SH) monomer into the 7.9 Å-resolution structure reveals that the sheath cylinder consists of axially stacked ß-hoops, each of which is comprised of two to six 400 nm-diameter rings of ß-strand arches (ß-rings). With both similarities to and differences from amyloid cross-ß fibril architecture, each ß-ring contains two giant ß-sheets contributed by ~ 450 SH monomers that entirely encircle the outer circumference of the cell. Tomograms of immature cells suggest models of sheath biogenesis: oligomerization of SH monomers into ß-ring precursors after their membrane-proximal cytoplasmic synthesis, followed by translocation through the unplugged end of a dividing cell, and insertion of nascent ß-hoops into the immature sheath cylinder at the junction of two daughter cells.

Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.

Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked "density scissors" connect with one another to form a "scissors stack network (SSN)" plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.

DSN/TdT recycling digestion based cyclic amplification strategy for microRNA assay.

Sensitive and specific detection of microRNAs (miRNAs) is of great significance for early cancer diagnosis. Here we report a simple and sensitive fluorescence signal amplification strategy that based on DSN/TdT recycling digestion for miRNA detection. DSN initiates DNA digestion on 3'-phosphate-primer/miRNA heteroduplex which causes miRNA recycle. The digested DNA strands with 3'-OH ends enable TdT to synthesize a polydeoxyguanylic tails on the 3'-end. The DNAs with polydeoxyguanylic tails are converted to double-stranded-DNA prior to initiation of DSN/TdT recycling digestion. With the cooperation of TdT and DSN, a new round of digestion and extension is triggered, leading to massive fluorophores separating and signal amplification. The amplification strategy produces large amounts of 3'-OH probes that can be used directly for dsDNA enrichment and DSN digestion. Moreover, both DSN digestion and TdT extension are sequence-independent reaction without the need of complex sequences design. In addition, this strategy is utilized to analyze miRNA samples from MCF-7 cell lysates and Cu (II) ion samples, indicating its potential application in actual sample analysis. The method shows a promising analytical platform for DNA nicking-related studies and tumor biomarkers measuring in clinical diagnostics.