Conformational Variability in Ground-State CFTR Lipoprotein Particle Cryo-EM Ensembles.

Cystic fibrosis transmembrane regulator (CFTR) is a dynamic membrane protein belonging to the ABC transporter family. It is unusual within this family as it is an ion channel, as opposed to a transporter. Activation of CFTR requires ATP and phosphorylation by PKA, and dysregulation of CFTR mediated salt and water homeostasis can lead to cystic fibrosis. Recent advancements in structural biological methods have led to more than 10 published CFTR structures, and, so far, all of these structures of CFTR, determined by cryo-EM, have been limited to detergent-purified protein preparations. To visualize CFTR in an environment that more closely represents its native membranous environment, we utilized two different lipoprotein particle encapsulation techniques: one in which the ion channel is first purified and then reconstituted using the membrane scaffolding protein Saposin A and another that uses the solubilizing polymer Sokalan CP9 (DIBMA) to extract CFTR directly from membranes. Structures derived from these types of preparations may better correlate to their function, for instance, the single-channel measurements from membrane vesicles.

High-speed high-resolution data collection on a 200 keV cryo-TEM.

Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (∼17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8-1.9 Šresolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 Šwas obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.

Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity.

A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine-threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.

Drug-Dependent Morphological Transitions in Spherical and Worm-Like Polymeric Micelles Define Stability and Pharmacological Performance of Micellar Drugs.

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy is not adequately addressed. This work addresses this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. It is observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. The role of different morphologies on pharmacological performance of drug loaded micelles against triple-negative breast cancer and pancreatic cancer tumor models is further evaluated. Spherical micelles accumulate rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulate more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug-micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

Structural basis of nucleosome-dependent cGAS inhibition.

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. By contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS uses two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome-binding interface exclusively occupies the strong double-stranded DNA (dsDNA)-binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions.

The structure of helical lipoprotein lipase reveals an unexpected twist in lipase storage.

Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.

Biological Applications at the Cutting Edge of Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Promotion of virus assembly and organization by the measles virus matrix protein.

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells.

UNLABELLED: Tetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes. IMPORTANCE: Tetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ∼15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications.

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.

Structure of glutaraldehyde cross-linked ryanodine receptor.

The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.