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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 ...

Gatan Inc.

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 β-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.

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

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.

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

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.

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

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 PDE6β subunits and two inhibitory PDE6γ subunits. Both PDE6α and PDE6β 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 (G). Recent cryo-electron microscopy structures of the PDE6αγβγ and PDE6αγβγ-(G) complexes have provided valuable knowledge shedding additional light on the allosteric activation of PDE6 by G. Here we discuss recent developments in our understanding of the mechanism of PDE6 activation.

Sahil Gulati

Gatan Inc.

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