Fitting small molecules to cryo-electron microscopy data

Recent innovations in the field of cryogenic-electron microscopy (cryo-EM) has enabled the visualisation of biological systems at atomic resolutions that rival that of X-ray crystallography. This is increasing the relevance of cryo-EM in the field of drug discovery, as it is now possible to solve high-resolution structures of biological complexes that may not have been amenable to crystallisation [1] and also in a more “native-like” state. However, it is not always possible to obtain structures to atomic resolutions with cryo-EM, currently only 16.28 % of structures deposited in the electron microscopy database [2] are at resolutions better than 3.0 Å, with the majority (45.05 %) at resolutions between 3.0 and 4.0 Å (correct as of December 2021). A vast body of work has been conducted with the aim of fitting biological macromolecules into cryo-EM at various resolutions [3–6]. However significantly less has been reported regarding the fitting of small molecules into cryo-EM maps. The work presented in this thesis aimed at developing methodologies that enable the fitting of small molecules to cryo-EM maps at resolutions from near atomic to 4.5Å. First, I used a fitting methodology that utilised consensus docking [7] in conjugation with a local difference mapping technique [8] to model the complex of the Eg5 kinesin motor domain with a novel inhibitor (GSK-1) in the presence of tubulin, into a 3.8 Å cryo-EM map (Chapter 2). The arrangement of structural elements within the protein allowed inferences to be made as to the mechanism of action of the drug [9]. Next, I present a new empirical molecular docking score for identifying correct ligand conformations within protein ligand complexes (Chapter 4). This score was integrated with goodness-of-fit scores commonly used for assessing the fit of biological molecules to cryo-EM maps [10]. Furthermore, we assessed the utility of this integrated score for fitting small molecules using simulated full maps and density difference maps (Chapter 4). This integrated score was then developed into a full methodology for fitting small molecules into cryo-EM maps, where its effectiveness was evaluated with experimental data at high (≤ 3.0 Å) and low (3.0 to 4.5 Å) resolution (Chapter 5). The accurate identification of protein ligand interactions from atomic models is an important consideration for drug discovery. To this end, a new software is presented that predicts protein ligand interactions using geometric parameters (Chapter 3). This software was benchmark using 35 high resolution protein-ligand complexes and compared to current state-of-the-art available software [11, 12]. Finally, I present the refined protein model of a Torpedo nicotinic acetylcholine receptor including the MX helix in a 6.6 Å cryo-EM map (Chapter 6). A combination of fitting software and bioinformatics identified the position of the MX helix relative to the cellular membrane. Our investigation suggested that the MX may function to entrap cholesterol, imposing rigidity to the receptor around the narrowest point of the central pore

Timeline Visualization Uncovers Gaps in Archived Tsunami Water Level Data

We demonstrate that data abstraction via a timeline visualization is highly effective at allowing one to discover patterns in the underlying data. We describe the rapid identification of data gaps in the archival time-series records of deep-ocean pressure and coastal water level observations collected to support the NOAA Tsunami Program and successful measures taken to rescue these data. These data gaps had persisted for years prior to the development of timeline visualizations to represent when data were collected. This approach can be easily extended to all types of time-series data and the author recommends this type of temporal visualization become a routine part of data management, whether one collects data or archives data.</p

Stoichiometry-Selective Antagonism of α4β2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4β2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4β2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)_{2}(β2)_{3} and (α4)_{3}(β2)_{2}, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4β2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4β2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)_{2}(β2)_{3} nAChRs than of (α4)_{3}(β2)_{2} nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)_{2}(β2)_{3} nAChRs with an IC_{50} of 26.4 ± 3.4 μM but displayed no significant inhibition of (α4)_{3}(β2)_{2} nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4β2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the β2(+)/β2(−) subunit interface, which is consistent with its selective inhibition of (α4)_{2}(β2)_{3}. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties

A benefit-cost analysis decision framework for mitigation of disease transmission at the wildlife–livestock interface

The economics of managing disease transmission at the wildlife–livestock interface have received heightened attention as agricultural and natural resource agencies struggle to tackle growing risks to animal health. In the fiscal landscape of increased scrutiny and shrinking budgets, resource managers seek to maximize the benefits and minimize the costs of disease mitigation efforts. To address this issue, a benefit-cost analysis decision framework was developed to help users make informed choices about whether and how to target disease management efforts in wildlife and livestock populations. Within the context of this framework, we examined the conclusions of a benefit-cost analysis conducted for vampire bat (Desmodus rotundus) rabies control in Mexico. The benefit-cost analysis decision framework provides a method that can be used to identify, assemble, and measure the components vital to the biological and economic efficiency of animal disease mitigation efforts. The framework can be applied to commercially-raised and free-ranging species at various levels of management – from detailed intervention strategies to broad programmatic actions. The ability of benefit cost analysis to illustrate the benefits of disease management projects per dollar spent allows for the determination of economic efficiency of alternative management actions. We believe this framework will be useful to the broader natural resource management community to maximize returns on financial and other resources invested in wildlife and livestock disease management programs

TEMPy2: a Python library with improved 3D electron microscopy density-fitting and validation workflows

Structural determination of molecular complexes by cryo-EM requires large, often complex processing of the image data that are initially obtained. Here, TEMPy2, an update of the TEMPy package to process, optimize and assess cryo-EM maps and the structures fitted to them, is described. New optimization routines, comprehensive automated checks and workflows to perform these tasks are described

Resting metabolic rate and lung function in wild offshore common bottlenose dolphins, Tursiops truncatus, near Bermuda

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Physiology 9 (2018): 886, doi:10.3389/fphys.2018.00886.Diving mammals have evolved a suite of physiological adaptations to manage respiratory gases during extended breath-hold dives. To test the hypothesis that offshore bottlenose dolphins have evolved physiological adaptations to improve their ability for extended deep dives and as protection for lung barotrauma, we investigated the lung function and respiratory physiology of four wild common bottlenose dolphins (Tursiops truncatus) near the island of Bermuda. We measured blood hematocrit (Hct, %), resting metabolic rate (RMR, l O2 ⋅ min-1), tidal volume (VT, l), respiratory frequency (fR, breaths ⋅ min-1), respiratory flow (l ⋅ min-1), and dynamic lung compliance (CL, l ⋅ cmH2O-1) in air and in water, and compared measurements with published results from coastal, shallow-diving dolphins. We found that offshore dolphins had greater Hct (56 ± 2%) compared to shallow-diving bottlenose dolphins (range: 30–49%), thus resulting in a greater O2 storage capacity and longer aerobic diving duration. Contrary to our hypothesis, the specific CL (sCL, 0.30 ± 0.12 cmH2O-1) was not different between populations. Neither the mass-specific RMR (3.0 ± 1.7 ml O2 ⋅ min-1 ⋅ kg-1) nor VT (23.0 ± 3.7 ml ⋅ kg-1) were different from coastal ecotype bottlenose dolphins, both in the wild and under managed care, suggesting that deep-diving dolphins do not have metabolic or respiratory adaptations that differ from the shallow-diving ecotypes. The lack of respiratory adaptations for deep diving further support the recently developed hypothesis that gas management in cetaceans is not entirely passive but governed by alteration in the ventilation-perfusion matching, which allows for selective gas exchange to protect against diving related problems such as decompression sickness.Funding for this project was provided by the Office of Naval Research (ONR YIP Award No. N000141410563, and Dolphin Quest, Inc. FHJ was supported by the Office of Naval Research (Award No. N00014-1410410) and an AIAS-COFUND fellowship from Aarhus Institute of Advanced Studies under the FP7 program of the EU (Agreement No. 609033)

A Social Marketing Intervention to Prevent Drowning Among Inner-City Youth

Water-related injuries and fatalities pose serious public health issues, especially to African American youth, a demographic group that drowns at disproportionately high rates. Aim. The purpose of this study was to determine if a social marketing intervention targeting the parents and guardians of inner-city youth (U.S. Midwest) could positively influence their perceptions concerning water safety. Method. Researchers employed a quasi-experimental design using matched pairs to evaluate the intervention. Participants consisted of parents who enrolled their children in a six-session survival-swimming course. Guided by the Health Belief Model, the researchers disseminated six prevention messages using six different channels (brochure, e-mail, SMS text message, postcard, Facebook, and window cling). Results. The findings from a two-way analysis of covariance revealed that treatment group participants’ knowledge and perceptions of water-related threat all changed favorably. Additionally, all participants planned to reenroll their children in swim lessons. Discussion. A social marketing campaign using the Health Belief Model improved inner-city parents’ knowledge regarding water safety and enhanced their self-efficacy. Conclusion. This study provides practitioners with feasible strategies (prevention messages) to supplement swim lessons, with the ultimate goal of preventing drowning among at-risk youth.https://doi-org.proxy-um.researchport.umd.edu/10.1177/152483991773255

New prediction categories in CASP15

Prediction categories in the Critical Assessment of Structure Prediction (CASP) experiments change with the need to address specific problems in structure modeling. In CASP15, four new prediction categories were introduced: RNA structure, ligand-protein complexes, accuracy of oligomeric structures and their interfaces, and ensembles of alternative conformations. This paper lists technical specifications for these categories and describes their integration in the CASP data management system

Widespread sampling biases in herbaria revealed from large-scale digitization

SUMMARY 1. Non-random collecting practices may bias conclusions drawn from analyses of herbarium records. Recent efforts to fully digitize and mobilize regional floras online offer a timely opportunity to assess commonalities and differences in herbarium sampling biases. 2. We determined spatial, temporal, trait, phylogenetic, and collector biases in ~5 million herbarium records, representing three of the most complete digitized floras of the world: Australia (AU), South Africa (SA), and New England, USA (NE). 3. We identified numerous shared and unique biases among these regions. Shared biases included specimens i) collected close to roads and herbaria; ii) collected more frequently during biological spring and summer; iii) of threatened species collected less frequently; and iv) of close relatives collected in similar numbers. Regional differences included i) over-representation of graminoids in SA and AU and of annuals in AU; and ii) peak collection during the 1910s in NE, 1980s in SA, and 1990s in AU. Finally, in all regions, a disproportionately large percentage of specimens were collected by very few individuals. We hypothesize that these mega-collectors, and along with their associated preferences and idiosyncrasies, shaped patterns of collection bias via ‘founder effects’. 4. Studies using herbarium collections should account for sampling biases, and future collecting efforts should avoid compounding these biases to the extent possible.Organismic and Evolutionary Biolog