Cryo-electron microscopy (cryoEM) techniques including single particle analysis (SPA), cryo-electron tomography (cryoET) and microcrystal electron diffraction (MED) has been widely used for multiple biological disciplines and successfully to reveal the molecular structures in a physiological environment. The key element for the successful application of cryoEM techniques to the individual research is highly dependent on the sample quality, performance of cryoEM and the reliable data processing pipeline. We have set up a unique cryoEM core facility in Keio University Bio2Q as an all-in-one lab and here we demonstrate the performance and entire pipeline of the structural analysis.

Transferrin receptor (TfR1) mediates iron uptake into cells via endocytosis at the blood brain barrier (BBB), making it a promising target molecule for BBB crossing strategies. We aimed to develop Nanobodies (NB) that show BBB permeability. Obtained NB (TO1) bound to mouse TfR1 with high affinity and its BBB permeability has been evaluated in vivo. However, TO1 showed almost no cross-reactivity against human TfR1. To elucidate the tight binding mechanism, the structure of the mouse TfR1-TO1 complex was solved at 2.22 Å resolution using single-particle analysis (SPA). We will discuss the detail molecular mechanism revealed by SPA.

The intracellular environment is highly crowded with macromolecules such as proteins and nucleic acids, and this molecular crowding critically influences biomolecular diffusion, interactions, reaction kinetics, and liquid–liquid phase separation. Despite its importance, quantitative and spatial visualization of molecular crowding in living cells remains challenging. In this presentation, we demonstrate that a fluorescent protein–fluorescent dye hybrid molecule, termed Chimera 1–TMR, in which tetramethylrhodamine (TMR) is conjugated to enhanced green fluorescent protein (EGFP) via a HaloTag, has the potential to function as a ratiometric fluorescent probe for imaging intracellular molecular crowding.

Pyrenoids are subcompartments of algal chloroplasts that concentrate Rubisco enzymes and their CO2 substrate, thereby increasing the efficiency of carbon fixation. Diatoms perform up to 20% of global CO2 fixation, but their pyrenoids remain poorly characterized at a molecular level. In vivo photo-crosslinking to catalogue components of diatom pyrenoids identified pyren2oid shell (PyShell) proteins localized to the pyrenoid periphery of both the pennate diatom, Pheaodactylum tricornutum, and the centric diatom, Thalassiosira pseudonana. In situ cryo-electron tomography (cryo-ET) revealed that the pyrenoids of both diatom species are encased in a lattice-like protein sheath. Disruption of PyShell expression in T. pseudonana resulted in the absence of this protein sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. Pyrenoids in mutant cells were fragmented and lacked the thylakoid membranes that normally traverse the Rubisco matrix, demonstrating how the PyShell plays a guiding role in establishing pyrenoid architecture. Recombinant PyShell proteins self-assembled into helical tubes and sheets, enabling us to determine a 2.4 Å-resolution PyShell structure. This in vitro structure was fitted into an in situ subtomogram average of the pyrenoid’s protein sheath (Fig.1), yielding a putative atomic model of the PyShell within diatom cells. The structure and function of the diatom PyShell provides a new molecular view of how CO is assimilated in the ocean, a crucial biome that is on the front lines of climate changen1). 

In recent years, cryo-electron microscopy (cryo-EM) has undergone a remarkable improvement in achievable resolution owing to the advent of direct electron detectors and advances in image processing methodologies. In contrast to X-ray crystallography, which requires crystallization, and NMR spectroscopy, which is limited by molecular size, cryo-EM enables structural analysis of large and heterogeneous macromolecular assemblies under near-native conditions. This advantage is particularly significant for samples exhibiting structural polymorphism or transient intermediate states, where cryo-EM has emerged as a powerful and often unique experimental approach.

In this study, we focused on protein folding processes, especially cotranslational folding that occurs during protein synthesis. The ribosome serves not only as a molecular machine for protein synthesis but also as the initial environment in which nascent polypeptide chains begin to fold. By exploiting the translational arrest sequence SecM, we stalled translation at defined positions and attempted to capture folding intermediates of nascent chains. Although the nascent polypeptide itself is small, the ribosome provides a sufficiently large scaffold to allow high-resolution structural analysis. Using this strategy, we successfully determined ribosome–nascent chain complexes at a resolution of 2.8 Å. The nascent chain within the ribosomal exit tunnel was clearly visualized, whereas regions exposed outside the tunnel became structurally heterogeneous and were not resolved in the averaged reconstruction.

We further extended our approach to the bacterial flagellum, a large self-assembling macromolecular machine. Flagellar assembly proceeds via tip growth, in which unfolded component proteins are transported through a narrow central channel and refold at the distal end. To visualize folding and assembly intermediates at the flagellar tip, we fused GFP to the hook protein FlgE. This strategy enabled us to determine the structure of a unique assembly intermediate in which a single FlgE molecule was polymerized, providing structural insight into an early stage of hook construction.

In this symposium, we will present the detailed structural features obtained from these two systems and discuss how cryo-EM can be applied to elucidate dynamic processes such as protein folding and self-assembly in large molecular machines.

Coming soon

Coming soon

Cryoelectron tomography (cryo-ET) is unique amongst structural biology techniques in its capability to visualize protein complexes, cytoskeletal structures, and organelle interactions at up to subnanometer resolution directly in their native cellular environment. This technique has therefore revolutionized our understanding of molecular architecture within cells. However, cryo-ET sample preparation, data acquisition, and downstream processing can be complex. This talk will therefore concentrate on the cryo-ET workflow, highlighting key technical challenges, possible solutions, and future directions of development.