leveraging-virtual-reality-for-immersive-segmentation-analysis-cryo

Leveraging Virtual Reality for Immersive Segmentation and Analysis of Cryo-Electron Tomography Data

Cryo-electron tomography (cryo-ET) is a powerful technique for visualizing the ultrastructure of cells in three dimensions (3D) at nanometer resolution. ...

Simons Electron Microscopy Center, New York Structural Biology Center

Columbia University

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

Architecture of the human erythrocyte ankyrin-1 complex.

Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the "Waffle Method" protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100-200 nm and dimensions of approximately 12 μm width and 15-20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h. Graphical abstract.

In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.

Square beams for optimal tiling in transmission electron microscopy.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

Recent advances in infectious disease research using cryo-electron tomography.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization.

Directed cell migration is driven by the front-back polarization of intracellular signalling. Receptor tyrosine kinases and other inputs activate local signals that trigger membrane protrusions at the front. Equally important is a long-range inhibitory mechanism that suppresses signalling at the back to prevent the formation of multiple fronts. However, the identity of this mechanism is unknown. Here we report that endoplasmic reticulum-plasma membrane (ER-PM) contact sites are polarized in single and collectively migrating cells. The increased density of these ER-PM contacts at the back provides the ER-resident PTP1B phosphatase more access to PM substrates, which confines receptor signalling to the front and directs cell migration. Polarization of the ER-PM contacts is due to microtubule-regulated polarization of the ER, with more RTN4-rich curved ER at the front and more CLIMP63-rich flattened ER at the back. The resulting ER curvature gradient leads to small and unstable ER-PM contacts only at the front. These contacts flow backwards and grow to large and stable contacts at the back to form the front-back ER-PM contact gradient. Together, our study suggests that the structural polarity mediated by ER-PM contact gradients polarizes cell signalling, directs cell migration and prolongs cell migration.

Endoplasmic reticulum-plasma membrane contact gradients direct cell migration.

Advances in cryo-electron tomography (cryo-ET) have produced new opportunities to visualize the structures of dynamic macromolecules in native cellular environments. While cryo-ET can reveal structures at molecular resolution, image processing algorithms remain a bottleneck in resolving the heterogeneity of biomolecular structures in situ. Here, we introduce cryoDRGN-ET for heterogeneous reconstruction of cryo-ET subtomograms. CryoDRGN-ET learns a deep generative model of three-dimensional density maps directly from subtomogram tilt-series images and can capture states diverse in both composition and conformation. We validate this approach by recovering the known translational states in Mycoplasma pneumoniae ribosomes in situ. We then perform cryo-ET on cryogenic focused ion beam-milled Saccharomyces cerevisiae cells. CryoDRGN-ET reveals the structural landscape of S. cerevisiae ribosomes during translation and captures continuous motions of fatty acid synthase complexes inside cells. This method is openly available in the cryoDRGN software.

Article	Year
Architecture of the human erythrocyte ankyrin-1 complex. Nature structural & molecular biology\| PubMed ID: 35835865	2022
In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method. Bio-protocol\| PubMed ID: 36618877	2022
Square beams for optimal tiling in transmission electron microscopy. Nature methods\| PubMed ID: 38238558	2024
Recent advances in infectious disease research using cryo-electron tomography. Frontiers in molecular biosciences\| PubMed ID: 38288336	2023
CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization. bioRxiv : the preprint server for biology\| PubMed ID: 38328036	2024
Endoplasmic reticulum-plasma membrane contact gradients direct cell migration. Nature\| PubMed ID: 38867038	2024
CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization. Research square\| PubMed ID: 39011111	2024
CryoDRGN-ET: deep reconstructing generative networks for visualizing dynamic biomolecules inside cells. Nature methods\| PubMed ID: 39025970	2024

Jake D. Johnston

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