Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

A cryo-ET survey of microtubules and intracellular compartments in mammalian axons

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.

In-cell structures of conserved supramolecular protein arrays at the mitochondria–cytoskeleton interface in mammalian sperm

Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

Cryo-electron tomography reveals structural insights into the membrane binding and remodeling activity of dynamin-like EHDs

Dynamin-related Eps15-homology domain containing proteins (EHDs) oligomerize on membrane surfaces into filaments leading to membrane remodeling. EHD crystal structures in an open and a closed conformation were previously reported, but structural information on the membrane-bound EHD oligomeric structure has remained enigmatic. Consequently, mechanistic insight into EHD-mediated membrane remodeling is lacking. Here, by using cryo-electron tomography and subtomogram averaging, we determined the structure of an EHD4 filament on a tubular membrane template at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests the AMPPNP-bound filament has a spontaneous curvature of approximately 1/70 nm-1. Combining the available structural and functional data, we propose a model of EHD-mediated membrane remodeling.

In situ cryo-ET structure of phycobilisome–photosystem II supercomplex from red alga

Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS–PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS–PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three ‘connector’ proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS–PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS–PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.

In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli

Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.

A flexible framework for multi-particle refinement in cryo-electron tomography

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo

Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line–based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography.

In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite’s apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.