High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.

Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a âˆ¼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a âˆ¼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining âˆ¼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.

Applying 3D ED/MicroED workflows toward the next frontiers.

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting-edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation.

High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.

Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these voltages, specifically a ~4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope. We also achieved a ~3.2 Å resolution reconstruction for a 115 kDa asymmetric protein complex, proving its effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ~3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, possibly bringing smaller sized particles within the scope of cryo-EM.

Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

New focus on regulation of the rod photoreceptor phosphodiesterase.

Rod photoreceptor phosphodiesterase (PDE6) is the key catalytic enzyme of visual phototransduction. PDE6 is the only member of the phosphodiesterase family that consists of a heterodimeric catalytic core composed of PDE6α and PDE6ß subunits and two inhibitory PDE6γ subunits. Both PDE6α and PDE6ß contain two regulatory GAF domains and one catalytic domain. GAF domains and the tightly bound PDE6γ subunits allosterically regulate the activity of the catalytic domain in association with the GTP-bound transducin alpha subunit (Gtα-GTP). Recent cryo-electron microscopy structures of the PDE6αγßγ and PDE6αγßγ-(Gtα-GTP)2 complexes have provided valuable knowledge shedding additional light on the allosteric activation of PDE6 by Gtα-GTP. Here we discuss recent developments in our understanding of the mechanism of PDE6 activation.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV.

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.

Melanopsin Carboxy-terminus phosphorylation plasticity and bulk negative charge, not strict site specificity, achieves phototransduction deactivation.

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and ß-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.

Climate change impacts and costs to U.S. electricity transmission and distribution infrastructure.

This study presents a screening-level analysis of the impacts of climate change on electricity transmission and distribution infrastructure of the U.S. In particular, the model identifies changes in performance and longevity of physical infrastructure such as power poles and transformers, and quantifies these impacts in economic terms. This analysis was evaluated for the contiguous U.S, using five general circulation models (GCMs) under two greenhouse gas emission scenarios, to analyze changes in damage and cost from the baseline period to the end of the century with three different adaptation strategies. Total infrastructure costs were found to rise considerably, with annual climate change expenditures increasing by as much as 25%. The results demonstrate that climate impacts will likely be substantial, though this analysis only captures a portion of the total potential impacts. A proactive adaptation strategy resulted in the expected costs of climate change being reduced by as much as 50% by 2090, compared to a scenario without adaptation. Impacts vary across the contiguous U.S. with the highest impacts in parts of the Southeast and Northwest. Improvements and extensions to this analysis would help better inform climate resiliency policies and utility-level planning for the future.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Stereospecific modulation of dimeric rhodopsin.

The classic concept that GPCRs function as monomers has been challenged by the emerging evidence of GPCR dimerization and oligomerization. Rhodopsin (Rh) is the only GPCR whose native oligomeric arrangement was revealed by atomic force microscopy demonstrating that Rh exists as a dimer. However, the role of Rh dimerization in retinal physiology is currently unknown. In this study, we identified econazole and sulconazole, two small molecules that disrupt Rh dimer contacts, by implementing a cell-based high-throughput screening assay. Racemic mixtures of identified lead compounds were separated and tested for their stereospecific binding to Rh using UV-visible spectroscopy and intrinsic fluorescence of tryptophan (Trp) 265 after illumination. By following the changes in UV-visible spectra and Trp265 fluorescence in vitro, we found that binding of R-econazole modulates the formation of Meta III and quenches the intrinsic fluorescence of Trp265. In addition, electrophysiological ex vivo recording revealed that R-econazole slows photoresponse kinetics, whereas S-econazole decreased the sensitivity of rods without effecting the kinetics. Thus, this study contributes new methodology to identify compounds that disrupt the dimerization of GPCRs in general and validates the first active compounds that disrupt the Rh dimer specifically.-Getter, T., Gulati, S., Zimmerman, R., Chen, Y., Vinberg, F., Palczewski, K. Stereospecific modulation of dimeric rhodopsin.

Estimates of Present and Future Asthma Emergency Department Visits Associated With Exposure to Oak, Birch, and Grass Pollen in the United States.

Pollen is an important environmental cause of allergic asthma episodes. Prior work has established a proof of concept for assessing projected climate change impacts on future oak pollen exposure and associated health impacts. This paper uses additional monitor data and epidemiologic functions to extend prior analyses, reporting new estimates of the current and projected future health burden of oak, birch, and grass pollen across the contiguous United States. Our results suggest that tree pollen in the spring currently accounts for between 25,000 and 50,000 pollen-related asthma emergency department (ED) visits annually (95% confidence interval: 14,000 to 100,000), roughly two thirds of which occur among people under age 18. Grass pollen in the summer season currently accounts for less than 10,000 cases annually (95% confidence interval: 4,000 to 16,000). Compared to a baseline with 21st century population growth but constant pollen, future temperature and precipitation show an increase in ED visits of 14% in 2090 for a higher greenhouse gas emissions scenario, but only 8% for a moderate emissions scenario, reflecting projected increases in pollen season length. Grass pollen, which is more sensitive to changes in climatic conditions, is a primary contributor to future ED visits, with the largest effects in the Northeast, Midwest, and Southern Great Plains regions. More complete assessment of the current and future health burden of pollen is limited by the availability of data on pollen types (e.g., ragweed), other health effects (e.g., other respiratory disease), and economic consequences (e.g., medication costs).

Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases.

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6αß2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αß heterodimer. Comparison of the PDE6αß2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

Specificity of the chromophore-binding site in human cone opsins.

The variable composition of the chromophore-binding pocket in visual receptors is essential for vision. The visual phototransduction starts with the cis-trans isomerization of the retinal chromophore upon absorption of photons. Despite sharing the common 11-cis-retinal chromophore, rod and cone photoreceptors possess distinct photochemical properties. Thus, a detailed molecular characterization of the chromophore-binding pocket of these receptors is critical to understanding the differences in the photochemistry of vision between rods and cones. Unlike for rhodopsin (Rh), the crystal structures of cone opsins remain to be determined. To obtain insights into the specific chromophore-protein interactions that govern spectral tuning in human visual pigments, here we harnessed the unique binding properties of 11-cis-6-membered-ring-retinal (11-cis-6mr-retinal) with human blue, green, and red cone opsins. To unravel the specificity of the chromophore-binding pocket of cone opsins, we applied 11-cis-6mr-retinal analog-binding analyses to human blue, green, and red cone opsins. Our results revealed that among the three cone opsins, only blue cone opsin can accommodate the 11-cis-6mr-retinal in its chromophore-binding pocket, resulting in the formation of a synthetic blue pigment (B6mr) that absorbs visible light. A combination of primary sequence alignment, molecular modeling, and mutagenesis experiments revealed the specific amino acid residue 6.48 (Tyr-262 in blue cone opsins and Trp-281 in green and red cone opsins) as a selectivity filter in human cone opsins. Altogether, the results of our study uncover the molecular basis underlying the binding selectivity of 11-cis-6mr-retinal to the cone opsins.

A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness.

The autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by mediating a GDP to GTP exchange in the Gα subunit. This leads to dissociation of the heterotrimer into Gα-GTP and Gßγ dimer. The Gα-GTP and Gßγ dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated Gßγ-signaling is a central element of various neurological and cancer-related anomalies. However, Gßγ also serves as a negative regulator of Gα that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Here we report a llama-derived nanobody (Nb5) that binds tightly to the Gßγ dimer. Nb5 responds to all combinations of ß-subtypes and γ-subtypes and competes with other Gßγ-regulatory proteins for a common binding site on the Gßγ dimer. Despite its inhibitory effect on Gßγ-mediated signaling, Nb5 has no effect on Gαq-mediated and Gαs-mediated signaling events in living cells.

Tumor stromal desmoplasia and inflammatory response uniquely predict survival with and without stratification for HPV tumor infection in OPSCC patients.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) positive for human papillomavirus (HPV) increases wolrd wide. AIMS/OBJECTIVES: The objective for this study has been to evaluate tumor phenotypes and tumor host responses with respect to five-year disease-specific survival (DSS) in HPV(+) and HPV(-) patients. MATERIAL AND METHODS: Two hundred patients with OPSCC have been treated between 1992 and 2010. Histopathology slides from these patients have been morphologically evaluated in formalin-fixed, paraffin-embedded (FFPE) stained with hematoxylin-eosin (HE). From HE-stained sections tumor phenotype (keratinization, fraction of mature cancer cells and pattern of invasion) and tumor host responses (inflammation and stromal desmoplasia) were evaluated with respect to five years DSS. RESULTS: High tumor inflammatory response and low stromal desmoplasia had an independent effect predicting better five-year DSS among all patients and when analyzed separately in the HPV(-) and HPV(+) cohort of patients using a Cox regression survival analysis that also included standard clinical prognostic variables among OPSCC patients. CONCLUSION: Tumor host responses, inflammation and stromal desmoplasia may become part of routine work-up in OPSCC patients due to prognostic value. SIGNIFICANCE: We present a method, accessible in most clinical locations and would give important additional information about prognosis in OPSCC patients.