Applications and prospects of cryo-EM in drug discovery.

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time- and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy (cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence (AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of medium-resolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.

Resting-state network complexity and magnitude changes in neonates with severe hypoxic ischemic encephalopathy.

Resting-state functional magnetic resonance imaging has revealed disrupted brain network connectivity in adults and teenagers with cerebral palsy. However, the specific brain networks implicated in neonatal cases remain poorly understood. In this study, we recruited 14 term-born infants with mild hypoxic ischemic encephalopathy and 14 term-born infants with severe hypoxic ischemic encephalopathy from Changzhou Children's Hospital, China. Resting-state functional magnetic resonance imaging data showed efficient small-world organization in whole-brain networks in both the mild and severe hypoxic ischemic encephalopathy groups. However, compared with the mild hypoxic ischemic encephalopathy group, the severe hypoxic ischemic encephalopathy group exhibited decreased local efficiency and a low clustering coefficient. The distribution of hub regions in the functional networks had fewer nodes in the severe hypoxic ischemic encephalopathy group compared with the mild hypoxic ischemic encephalopathy group. Moreover, nodal efficiency was reduced in the left rolandic operculum, left supramarginal gyrus, bilateral superior temporal gyrus, and right middle temporal gyrus. These results suggest that the topological structure of the resting state functional network in children with severe hypoxic ischemic encephalopathy is clearly distinct from that in children with mild hypoxic ischemic encephalopathy, and may be associated with impaired language, motion, and cognition. These data indicate that it may be possible to make early predictions regarding brain development in children with severe hypoxic ischemic encephalopathy, enabling early interventions targeting brain function. This study was approved by the Regional Ethics Review Boards of the Changzhou Children's Hospital (approval No. 2013-001) on January 31, 2013. Informed consent was obtained from the family members of the children. The trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR1800016409) and the protocol version is 1.0.

Uncoating Mechanism of Carnation Mottle Virus Revealed by Cryo-EM Single Particle Analysis.

Genome uncoating is a prerequisite for the successful infection of plant viruses in host plants. Thus far, little is known about the genome uncoating of the Carnation mottle virus (CarMV). Here, we obtained two reconstructions of CarMV at pH7 in the presence (Ca-pH7) and absence (EDTA-pH7) of calcium ions by Cryo-EM single particle analysis, which achieved 6.4 Å and 8 Å resolutions respectively. Our results showed that chelation of the calcium ions under EDTA-pH7 resulted in reduced interaction between the subunits near the center of the asymmetric unit but not overall size change of the viral particles, which indicated that the role of the calcium ions in CarMV was not predominantly for the structural preservation. Part of the genomic RNA closest to the capsid was found to be located near the center of the asymmetric unit, which might result from the interaction between genomic RNA and Lys194 residues. Together with the electrostatic potential analysis on the inner surface of the asymmetric unit, the reduced interaction near the center of the asymmetric unit under EDTA-pH7 suggested that the genome release of CarMV might be realized through the center of the asymmetric unit.

Insight into the three-dimensional structure of maize chlorotic mottle virus revealed by Cryo-EM single particle analysis.

Maize chlorotic mottle virus (MCMV) is the only member of the Machlomovirus genus in the family Tombusviridae. Here, we obtained the Cryo-EM structure of MCMV by single particle analysis with most local resolution at approximately 4 Å. The Cα backbone was built based on residues with bulky side chains. The resolved C-terminus of the capsid protein subunit and obvious openings at the 2-fold axis demonstrated the compactness of the asymmetric unit, which indicates an important role in the stability of MCMV. The Asp116 residue from each subunit around the 5-fold and 3-fold axes contributed to the negative charges in the centers of the pentamers and hexamers, which might serve as a solid barrier against the leakage of genomic RNA. Finally, the loops most exposed on the surface were analyzed and are proposed to be potential functional sites related to MCMV transmission.

5-aminolevulinic acid-mediated photodynamic therapy on cervical condylomata acuminata.

OBJECTIVE: The aim of this study was to evaluate the efficacy of 5-aminolevulinic acid-mediated photodynamic therapy (PDT) on cervical condylomata accuminate (CA) and to determine the ultrastructural changes of the lesion after treatment. BACKGROUND DATA: Although there are many methods to treat cervical CA, a high recurrence rate and cervical scars are troublesome problems. MATERIALS AND METHODS: Forty-eight patients with cervical CA were treated with topical 20% ALA gel followed by PDT through a cylindrical fiber with a wavelength of 630 nm of laser light. The treatment was repeated 7 days later if the lesion had not been removed completely. Eight cases were biopsied before and after treatment, and the biopsied lesions were evaluated by electron microscopy. RESULTS: After the first PDT treatment, the cervical condylomatous lesions were removed completely in 62.5% (30/48). The complete response (CR) rate was 95.8% (46/48) after three treatments. The recurrence rate was 4.4% at the 12-month follow-up. Electron microscopy revealed that ALA-PDT damaged proliferative keratinocytes of CA lesions while the surrounding normal tissue injury was less damaged. CONCLUSIONS: ALA-PDT is an effective and minimally invasive treatment for cervical CA.

The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila.

BACKGROUND: Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. RESULTS: In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. CONCLUSIONS: The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

Three-dimensional display and arbitrary region interactive segmentation of high-resolution virus capsids from cryo-electron microscopy single particle reconstruction.

Recent advances in cryo-electron microscopy (cryo-EM) instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3-D) structure determination of virus. In order to visualize and effectively understand the 3-D structure, we present a display method based on surface rendering, which has the function of 3-D arbitrary region interactive segmentation and quantitative analysis, and integrate them into a software package called CEM-3DVDSS (cryo-EM 3-D virus display and arbitrary region segmentation system). CEM-3DVDSS consists of a complete set of modular programs for 3-D display and segmentation of icosahedral virus, which is organized under a graphical user interface and provides user-friendly options. First, we convert volume data in the MRC format obtained by cryo-EM single particle reconstruction to the format of our own software; in the preprocessing step, the original volume data are compressed and a better vector dimension is found for controlling the speed and detail of display. Then, the new volume data can be displayed and segmented using CEM-3DVDSS. We demonstrate the applicability of CEM-3DVDSS by displaying the 3-D structures of 2.5 nm (resolution) BmCPV (Bombyx mori cytoplasmic polyhedrosis virus), 2.5 nm CSBV (Chinese Sacbrood bee virus) and 1.4 nm C6/36DNV (Densonucleosis virus). As a result, both the 3-D display speed and signal-to-noise ratio of CEM-3DVDSS are improved compared with the original method, and the segmentation results become precise and more intact with additional function of quantitative analysis of 3-D structure.

Morphology and morphogenesis of severe acute respiratory syndrome (SARS)-associated virus.

After infecting the Vero E6 cells by nasal/throat swabs collected from SARS patients, we studied the SARS-associated virus by electron microscopy and molecular biological technique. The results show that the diameter of newly isolated virus is about 50 nm without envelope or 100 nm with envelope. The virus was proved to be a new coronavirus by RT-PCR and it responded positively to convalescent-phase serum specimen from SARS patients, which is the evidence that this new virus is etiologically linked to the outbreak of SARS. The morphogenesis and distribution of the virus are also discussed in this article.

Structure of Cytoplasmic Polyhedrosis Virus from Bombyx mori.

The structures of full and empty capsids of CPV were studied by negative staining and electron cryomicroscopy and computer reconstruction techniques. By comparing the structures and biochemical compositions, the CPV was identified as a single-layered capsid with its five structural proteins located on it. This single capsid is arranged according to T=1 icosahedral symmetry with 12 turret-like spikes at its icosahedral vertices. The empty and full CPV show identical capsid but differ inside. The dense and ordered genomic dsRNA is located inside the full CPV. The internal space of the empty CPV has almost no electron density except for 12 electron densities attributed the transcriptional enzyme complexes extending inward from the base part of CPV spikes.