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Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.

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Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7 Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.

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The members of the superfamily of Transient Receptor Potential (TRP) ion channels are physiologically important molecules that have been studied for many years and are still being intensively researched. Among the vanilloid TRP subfamily, the TRPV4 ion channel is an interesting protein due to its involvement in several essential physiological processes and in the development of various diseases. As in other proteins, changes in its function that lead to the development of pathological states, have been closely associated with modification of its regulation by different molecules, but also by the appearance of mutations which affect the structure and gating of the channel. In the last few years, some structures for the TRPV4 channel have been solved. Due to the importance of this protein in physiology, here we discuss the recent progress in determining the structure of the TRPV4 channel, which has been achieved in three species of animals (Xenopus tropicalis, Mus musculus, and Homo sapiens), highlighting conserved features as well as key differences among them and emphasizing the binding sites for some ligands that play crucial roles in its regulation.

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Viral diseases are expected to cause new epidemics in the future, therefore, it is essential to assess how viral diversity is represented in terms of deposited protein structures. Here, data were collected from the Protein Data Bank to screen the available structures of viruses of interest to WHO. Excluding SARS-CoV-2 and HIV-1, less than 50 structures were found per year, indicating a lack of diversity. Efforts to determine viral structures are needed to increase preparedness for future public health challenges.

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The solute carrier family 12A (SLC12A) superfamily of membrane transporters modulates the movement of cations coupled with chloride across the membrane. In doing so, these cotransporters are involved in numerous aspects of human physiology: cell volume regulation, ion homeostasis, blood pressure regulation, and neurological action potential via intracellular chloride concentration modulation. Their physiological characterization has been largely studied; however, understanding the mechanics of their function and the relevance of structural domains or specific amino acids has been a pending task. In recent years, single-particle cryogenic electron microscopy (cryo-EM) has been successfully applied to members of the SLC12A family including all K+:Cl- cotransporters (KCCs), Na+:K+:2Cl- cotransporter NKCC1, and recently Na+:Cl- cotransporter (NCC); revealing structural elements that play key roles in their function. The present review analyzes the data provided by these cryo-EM reports focusing on structural domains and specific amino acids involved in ion binding, domain interactions, and other important SCL12A structural elements. A comparison of cryo-EM data from NKCC1 and KCCs is presented in the light of the two recent NCC cryo-EM studies, to propose insight into structural elements that might also be found in NCC and are necessary for its proper function. In the final sections, the importance of key coordination residues for substrate specificity and their implication on various pathophysiological conditions and genetic disorders is reviewed, as this could provide the basis to correlate structural elements with the development of novel and selective treatments, as well as mechanistic insight into the function and regulation of cation-coupled chloride cotransporters (CCCs).

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Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21 years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

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Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing 3D density maps of biomolecules at near-atomic resolution. However, map validation is still an open issue. Despite several efforts from the community, it is possible to overfit 3D maps to noisy data. Here, we develop a novel methodology that uses a small independent particle set (not used during the 3D refinement) to validate the maps. The main idea is to monitor how the map probability evolves over the control set during the 3D refinement. The method is complementary to the gold-standard procedure, which generates two reconstructions at each iteration. We low-pass filter the two reconstructions for different frequency cutoffs, and we calculate the probability of each filtered map given the control set. For high-quality maps, the probability should increase as a function of the frequency cutoff and the refinement iteration. We also compute the similarity between the densities of probability distributions of the two reconstructions. As higher frequencies are included, the distributions become more dissimilar. We optimized the BioEM package to perform these calculations, and tested it over systems ranging from quality data to pure noise. Our results show that with our methodology, it possible to discriminate datasets that are constructed from noise particles. We conclude that validation against a control particle set provides a powerful tool to assess the quality of cryo-EM maps.

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The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Šresolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

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Segmentation is one of the most important stages in the 3D reconstruction of macromolecule structures in cryo-electron microscopy. Due to the variability of macromolecules and the low signal-to-noise ratio of the structures present, there is no generally satisfactory solution to this process. This work proposes a new unsupervised particle picking and segmentation algorithm based on the composition of two well-known image filters: Anisotropic (Perona-Malik) diffusion and non-negative matrix factorization. This study focused on keyhole limpet hemocyanin (KLH) macromolecules which offer both a top view and a side view. Our proposal was able to detect both types of views and separate them automatically. In our experiments, we used 30 images from the KLH dataset of 680 positive classified regions. The true positive rate was 95.1% for top views and 77.8% for side views. The false negative rate was 14.3%. Although the false positive rate was high at 21.8%, it can be lowered with a supervised classification technique.

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The pentameric γ-aminobutyric acid type A receptors are ion channels activated by ligands, which intervene in the rapid inhibitory transmission in the mammalian CNS. Due to their rich pharmacology and therapeutic potential, it is essential to understand their structure and function thoroughly. This deep characterization was hampered by the lack of experimental structural information for many years. Thus, computational techniques have been extensively combined with experimental data, in order to undertake the study of γ-aminobutyric acid type A receptors and their interaction with drugs. Here, we review the exciting journey made to assess the structures of these receptors and outline major outcomes. Finally, we discuss the brand new structure of the α1ß2γ2 subtype and the amazing advances it brings to the field.

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Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.

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UNLABELLED: The purpose of this work was to investigate the susceptibility of an aluminum adjuvant and an aluminum-adjuvanted native outer membrane vesicle (nOMV) vaccine formulation to freeze/thaw-induced agglomeration using static light scattering and micro-flow Imaging analysis; and to evaluate the use of propylene glycol as a vaccine formulation excipient by which freeze/thaw-induced agglomeration of a nOMV vaccine formulation could be mitigated. Our results indicate that including 7% v/v propylene glycol in an nOMV containing aluminum adjuvanted vaccine formulation, mitigates freeze/thaw-induced agglomeration. LAY ABSTRACT: We evaluated the effect of freeze-thawing on an aluminum adjuvant and an aluminum adjuvanted native outer membrane vesicle (nOMV) vaccine formulation. Specifically, we characterized the freeze/thaw-induced agglomeration through the use of static light scattering, micro-flow imaging, and cryo-electron microscopy analysis. Further, we evaluated the use of 0-9% v/v propylene glycol as an excipient which could be included in the formulation for the purpose of mitigating the agglomeration induced by freeze/thaw. The results indicate that using 7% v/v propylene glycol as a formulation excipient is effective at mitigating agglomeration of the nOMV vaccine formulation, otherwise induced by freeze-thawing.

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