Proteins by and large carry out their molecular functions in a folded state when residues, distant in sequence, assemble together in 3D space to bind a ligand, catalyze a reaction, form a channel, or exert another concerted macromolecular interaction. It has been long recognized that covariance of amino acids between distant positions within a protein sequence allows for the inference of long range contacts to facilitate 3D structure modeling. In this work, we investigated whether covariance analysis may reveal residues involved in the same molecular function. Building upon our previous work, CoeViz, we have conducted a large scale covariance analysis among 7,595 non-redundant proteins with resolved 3D structures to assess 1) whether the residues with the same function coevolve, 2) which covariance metric captures such couplings better, and 3) how different molecular functions compare in this context. We found that the chi-squared metric is the most informative for the identification of coevolving functional sites, followed by the Pearson correlation-based, whereas mutual information is the least informative. Of the seven categories of the most common natural ligands, including coenzyme A, dinucleotide, DNA/RNA, heme, metal, nucleoside, and sugar, the trace metal binding residues display the most prominent coupling, followed by the sugar binding sites. We also developed a web-based tool, CoeViz 2, that enables the interactive visualization of covarying residues as cliques from a larger protein graph. CoeViz 2 is publicly available at https://research.cchmc.org/CoevLab/.
Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. While the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity (https://research.cchmc.org/Ciliome_Gene_Expression/). Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by utilizing Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore long-standing questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.
Small molecule docking and virtual screening of candidate compounds have become an integral part of drug discovery pipelines, complementing and streamlining experimental efforts in that regard. In this chapter, we describe specific software packages and protocols that can be used to efficiently set up a computational screening using a library of compounds and a docking program. We also discuss consensus- and clustering-based approaches that can be used to assess the results, and potentially re-rank the hits. While docking programs share many common features, they may require tailored implementation of virtual screening pipelines for specific computing platforms. Here, we primarily focus on solutions for several public domain packages that are widely used in the context of drug development.
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