Cationic Amino Acid Identity and Net Charge Influence Condensate Properties in .

Biomolecular condensates (BMCs) are central to subcellular organization, influencing processes from RNA metabolism to the stress response and amyloid pathologies. Despite their near ubiquity, we still do not fully understand how the primary sequence of biomolecules influences the formation and dynamics of condensates. Here, we examine how cationic amino acid identity shapes the properties of protein-RNA coacervates.

Overcoming air-water interface-induced artifacts in Cryo-EM with protein nanocrates.

Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as "nanocrates" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).

CryoDRGN-AI: neural ab initio reconstruction of challenging cryo-EM and cryo-ET datasets.

Proteins and other biomolecules form dynamic macromolecular machines that are tightly orchestrated to move, bind and perform chemistry. Cryo-electron microscopy and cryo-electron tomography can access the intrinsic heterogeneity of these complexes and are therefore key tools for understanding their function. However, three-dimensional reconstruction of the collected imaging data presents a challenging computational problem, especially without any starting information, a setting termed ab initio reconstruction.

Cationic amino acid identity and net charge influence condensate properties in .

Understanding the formation of biomolecular condensates (BMC) in biological systems has proven to be a paradigm shift in our understanding of the subcellular organization of biomacromolecules. From RNA metabolism, stress response mechanisms, and amyloidogenic pathologies, condensates have been implicated to play a role in a myriad of cellular phenomena. Despite their near ubiquity, we still do no wholly understand how the primary sequence of biomolecules influences their biophysical and rheological properties.

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. However, the manual segmentation of cellular components in cryo-ET data remains a significant bottleneck due to its complexity and time-consuming nature. In this work, we present a novel segmentation workflow that integrates advanced virtual reality (VR) software to enhance both the efficiency and accuracy of segmenting cryo-ET datasets.

CryoDRGN-ET: deep reconstructing generative networks for visualizing dynamic biomolecules inside cells.

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.

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

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.

Endoplasmic reticulum-plasma membrane contact gradients direct cell migration.

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.

CryoDRGN-AI: Neural reconstruction of challenging cryo-EM and cryo-ET datasets.

Proteins and other biomolecules form dynamic macromolecular machines that are tightly orchestrated to move, bind, and perform chemistry. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) can access the intrinsic heterogeneity of these complexes and are therefore key tools for understanding their function. However, 3D reconstruction of the collected imaging data presents a challenging computational problem, especially without any starting information, a setting termed reconstruction.

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

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.

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

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.

Square beams for optimal tiling in transmission electron microscopy.

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.

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

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.

Architecture of the human erythrocyte ankyrin-1 complex.

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.