Biology subjects

Williams, C. J.

Publications and source records attributed to Williams, C. J..

17 recordsLinked to original sources

bioRxiv2026

Neonatal diethylstilbestrol exposure disrupts uterine epithelial apical-basal polarity and partial EMT state

The developing female reproductive tract is highly sensitive to external hormonal stimulation, which can result in infertility and gynecologic diseases. To determine the underlying mechanisms, we used a mouse model to test the immediate, cell type-specific effects of neonatal exposure to the estrogenic chemical, diethylstilbestrol (DES), on the developing uterus. We found that control uterine epithelium is in a partial epithelial-mesenchymal transition state that is lost following DES exposure. This is accompanied by evidence of premature differentiation including altered apical-basal cell polarity and absence of the Lgr5+ epithelial stem cell population required for uterine gland formation. Cell-cell communication between epithelial and mesenchymal cells is restructured, and Wnt signaling is persistently reduced. The DES-exposed uterine mesenchyme has early signs of fibrosis through increased deposition of extracellular matrix collagen.Mechanistically, DES exposure causes cell type-specific changes in chromatin accessibility and gene expression, most prominently in epithelial cells. These changes can be explained in part by cell-specific alterations in chromatin looping at enhancer regions in concert with alterations in ER binding. These findings suggest that reprogramming cell type-specific differentiation trajectories and extracellular matrix characteristics underlie the long-term phenotypic effects of developmental exposure to estrogenic endocrine disrupting chemicals. These changes lead to functional impairment of adult tissues and increased cancer risk. Significance StatementUterine development is strongly impacted by brief exposure to estrogenic endocrine disruptors, but it is unclear why development is such a sensitive time point. This study employed multiomic analysis to identify cell type-specific uterine developmental trajectories in neonatal mice exposed to the estrogenic chemical, diethylstilbestrol, and compared these to controls. Control epithelium was under the influence of carefully orchestrated Wnt/{beta}-catenin signaling and was in a partial epithelial-to-mesenchymal transition state. DES exposure repressed Wnt/{beta}-catenin signaling and drove the epithelium toward full differentiation, resulting in the loss of both epithelial stem cells and normal apical-basal polarity. These changes provide an explanation for how endocrine disruptors can divert intrinsically programmed developmental trajectories to alter adult organ function.

developmental biology↗

bioRxiv2025

Categorizing prediction modes within low-pLDDT regions of AlphaFold2 structures

AlphaFold2 protein structure predictions are widely available for structural biology uses. These predictions, especially for eukaryotic proteins, frequently contain extensive regions predicted below the pLDDT 70 level, the rule-of-thumb cutoff for high confidence. This work identifies major modes of behavior within low-pLDDT regions through a survey of human proteome predictions provided by the AlphaFold Protein Structure Database. The near-predictive mode resembles folded protein and can be a nearly accurate prediction. Barbed wire is extremely unproteinlike, being recognized by wide looping coils, an absence of packing contacts, and numerous signature validation outliers, and it likely represents a nonpredicted region. Pseudostructure presents an intermediate behavior with a misleading appearance of isolated and badly formed secondary structure-like elements. These prediction modes are compared with annotations of disorder from MobiDB, showing general correlation between barbed wire/pseudostructure and many measures of disorder, an association between pseudostructure and signal peptides, and an association between near-predictive and regions of conditional folding. To enable users to identify these regions within a prediction, a new Phenix tool is developed encompassing the results of this work, including prediction annotation, visual markup, and residue selection based on these prediction modes. This tool will help users develop expertise in interpreting difficult AlphaFold predictions and identify the near-predictive regions that can aid in molecular replacement when a prediction does not contain enough high-pLDDT regions.

biochemistry↗

bioRxiv2025

Calcium signals shape metabolic control of H3K27ac and H3K18la to regulate EGA

The use of assisted reproductive technologies (ART) has enabled the birth of over 9 million babies; but it is associated with increased risks of negative metabolic outcomes in offspring. Yet, the underlying mechanism remains unknown. Calcium (Ca2+) signals, which initiate embryo development at fertilization, are frequently disrupted in human ART. In mice, abnormal Ca2+ signals at fertilization impair embryo development and adult offspring metabolism. Changes in intracellular Ca2+ drive mitochondrial activity and production of metabolites used by the epigenetic machinery. For example, acetyl-CoA (derived mainly from pyruvate) and lactyl-CoA (derived from lactate) are used for writing H3K27ac and H3K18la marks that orchestrate initiation of development. Using both a genetic mouse model and treatment with ionomycin to raise intracellular Ca2+ of wild-type fertilized eggs, we found that excess Ca2+ at fertilization changes metabolic substrate availability, causing epigenetic changes that impact embryo development and offspring health. Specifically, increased Ca2+ exposure at fertilization led to increased H3K27ac levels and decreased H3K18la levels at the 1-cell (1C) stage, that persisted until the 2-cell (2C) stage. Ultralow input CUT&Tag revealed significant differences in H3K27ac and H3K18la genomic profiles between control and ionomycin groups. In addition, increased Ca2+ exposure resulted in a marked reduction in global transcription at the 1C stage that persisted through the 2C stage due to diminished activity of RNA polymerase I. Excess Ca2+ following fertilization increased pyruvate dehydrogenase activity (enzyme that converts pyruvate to acetyl-CoA) and decreased total lactate levels. Provision of exogenous lactyl-CoA before ionomycin treatment restored H3K18la levels at the 1C and 2C stages and rescued global transcription to control levels. Our findings demonstrate conclusively that Ca2+ dynamics drive metabolic regulation of epigenetic reprogramming at fertilization and alter EGA.

developmental biology↗

bioRxiv2025

RNAprecis: Prediction of full-detail RNA conformation from the experimentally best-observed sparse parameters

We address the problem of predicting high-detail RNA structure geometry from the information available in low-detail experimental maps. Here, low-detail refers to resolutions {approx} 2.5-3.5[A], where the location of the phosphate groups and the glycosidic bonds can be determined from experimental maps but all other backbone atom positions cannot. In contrast, higher-resolution maps allow high-detail determinations of all backbone atomic positions. To this end, we first create a gold standard dataset of highly curated, experimentally supported RNA suites. Second, we develop and employ a modified version of the previously devised algorithm MINT-AGE to learn clusters that are in high correspondence with the gold standards conformational classes of suites based on 3D RNA structure. Since some of the gold standard classes are of very small size, a new modified version of MINT-AGE is able to also identify very small clusters. Third, we create a new conformer prediction algorithm, RNAprecis, which assigns low-detail structures to newly designed 3D shape coordinates. Our improvements include: (i) learned classes augmented to cover also very low sample sizes and (ii) replacing distances from clusters by Bayesian posterior probabilities. On test data containing suites modeled as conformational outliers, RNAprecis shows good results suggesting that our learning method generalizes well. In particular, we show that the modified MINT-AGE clustering can more finely delineate between previously unseen suite conformer separations. For example, the 0a conformer has been separated into two clusters seen in different structural contexts. Such new distinctions can have implications for biochemical interpretation of RNA structure.

molecular biology↗

bioRxiv2024

Structural Convergence and Water-Mediated Substrate Mimicry Enable Broad Neuraminidase Inhibition by Human Antibodies

Influenza has been responsible for multiple global pandemics and seasonal epidemics and claimed millions of lives. The imminent threat of a panzootic outbreak of avian influenza H5N1 virus underscores the urgent need for pandemic preparedness and effective countermeasures, including monoclonal antibodies (mAbs). Here, we characterize human mAbs that target the highly conserved catalytic site of viral neuraminidase (NA), termed NCS mAbs, and the molecular basis of their broad specificity. Cross-reactive NA-specific B cells were isolated by using stabilized NA probes of non-circulating subtypes. We found that NCS mAbs recognized multiple NAs of influenza A as well as influenza B NAs and conferred prophylactic protections in mice against H1N1, H5N1, and influenza B viruses. Cryo-electron microscopy structures of two NCS mAbs revealed that they rely on structural mimicry of sialic acid, the substrate of NA, by coordinating not only amino acid side chains but also water molecules, enabling inhibition of NA activity across multiple influenza A and B viruses, including avian influenza H5N1 clade 2.3.4.4b viruses. Our results provide a molecular basis for the broad reactivity and inhibitory activity of NCS mAbs targeting the catalytic site of NA through substrate mimicry.

immunology↗

bioRxiv2024

The Polygenic Score - Rare Variant Causal Pivot: A Conditional Approach to Discovery in Complex Disease Genetics

We present the Causal Pivot (CP) as a structural causal model (SCM) for analyzing genetic heterogeneity in complex diseases. The CP leverages one established causal factor to detect the contribution of a second suspected cause. Specifically, polygenic risk scores (PRS) serve as known causes, while rare variants (RV) or RV ensembles are evaluated as candidate causes. The CP incorporates outcome-induced association by conditioning on disease status. We derive a conditional maximum likelihood procedure for binary and quantitative traits and develop the Causal Pivot Likelihood Ratio Test (CP-LRT) to detect causal signals. Through simulations, we demonstrate the CP-LRTs robust power and superior error control compared to alternatives. We apply the CP-LRT to UK Biobank (UKB) data, analyzing three exemplar diseases: hypercholesterolemia (HC, LDL-c [≥] 4.9 mmol/L; nc=24,656), breast cancer (BC, ICD10 C50; nc=12,479), and Parkinsons disease (PD, ICD10 G20; nc=2,940). For PRS, we utilize UKB-derived values, and for RVs, we analyze ClinVar pathogenic/likely pathogenic variants and loss-of-function mutations in disease-relevant genes: LDLR for HC, BRCA1 for BC, and GBA for PD. Significant CP-LRT signals were detected for all three diseases. Cross-disease and synonymous variant analyses serve as controls. We further develop ancestry adjustment using matching and inverse probability weighting, and we extend the CP to examine oligogenic burden in the lysosomal storage pathway for PD. The CP reveals an approach to address heterogeneity and is an extensible method for inference and discovery in complex disease genetics.

genetics↗

bioRxiv2024

Estrogen receptor alpha mediated repression of PRICKLE1 destabilizes REST and promotes uterine fibroid pathogenesis

Uterine fibroids (leiomyomas), benign tumors of the myometrial smooth muscle layer, are present in over 75% of women, often causing severe pain, menorrhagia and reproductive dysfunction. The molecular pathogenesis of fibroids is poorly understood. We previously showed that the loss of REST (RE-1 Silencing Transcription factor), a tumor suppressor, in fibroids leads to activation of PI3K/AKT-mTOR pathway. We report here a critical link between estrogen receptor alpha (ER) and the loss of REST, via PRICKLE1. PRICKLE1 expression is markedly lower in leiomyomas, and the suppression of PRICKLE1 significantly down regulates REST protein levels. Conversely, overexpression of PRICKLE1 resulted in the restoration of REST in cultured primary leiomyoma smooth muscle cells (LSMCs). Crucially, mice exposed neonatally to environmental estrogens, proven risk factors for fibroids, expressed lower levels of PRICKLE1 and REST in the myometrium. Using mice that lack either endogenous estrogen (Lhb-/- mice) or ER (Esr1-/- mice), we demonstrate that Prickle1 expression in the myometrium is suppressed by estrogen through ER. Enhancer of zeste homolog 2 (EZH2) is known to participate in the repression of specific ER target genes. Uterine leiomyomas express increased levels of EZH2 that inversely correlate with the expression of PRICKLE1. Using chromatin immunoprecipitation, we provide evidence for association of EZH2 with the PRICKLE1 promoter and for hypermethylation of H3K27 within the regulatory region of PRICKLE1 in leiomyomas. Additionally, siRNA mediated knockdown of EZH2 leads to restoration of PRICKLE1 in LSMCs. Collectively, our results identify a novel link between estrogen exposure and PRICKLE1/REST-regulated tumorigenic pathways in leiomyomas.

cell biology↗

bioRxiv2022

Dynamic DNA methylation turnover in gene bodies is associated with enhanced gene expression plasticity

BackgroundIn several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance has remained unclear. ResultsWe report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity. ConclusionsThis study proposes a new model for GbM in regulating gene expression plasticity, including a newly discovered type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.

genomics↗

bioRxiv2022

AlphaFold predictions: great hypotheses but no match for experiment

AI-based methods such as AlphaFold have revolutionized structural biology, often making it possible to predict protein structures with high accuracy. The accuracies of these predictions vary, however, and they do not include ligands, covalent modifications or other environmental factors. Here we focus on very-high-confidence parts of AlphaFold predictions, evaluating how well they can be expected to describe the structure of a protein in a particular environment. We compare predictions with experimental crystallographic maps of the same proteins for 102 crystal structures. In many cases, those parts of AlphaFold predictions that were predicted with very high confidence matched experimental maps remarkably closely. In other cases, these predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. Overall, C atoms in very-high-confidence parts of AlphaFold predictions differed from corresponding crystal structures by a median of 0.6 [A], and about 10% of these differed by more than 2 [A], each about twice the values found for pairs of crystal structures containing the same components but determined in different space groups. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.

biochemistry↗

bioRxiv2022

Accelerating crystal structure determination with iterative AlphaFold prediction

Experimental structure determination can be accelerated with AI-based structure prediction methods such as AlphaFold. Here we present an automatic procedure requiring only sequence information and crystallographic data that uses AlphaFold predictions to produce an electron density map and a structural model. Iterating through cycles of structure prediction is a key element of our procedure: a predicted model rebuilt in one cycle is used as a template for prediction in the next cycle. We applied this procedure to X-ray data for 215 structures released by the Protein Data Bank in a recent 6-month period. In 87% of cases our procedure yielded a model with at least 50% of C atoms matching those in the deposited models within 2[A]. Predictions from our iterative template-guided prediction procedure were more accurate than those obtained without templates. We suggest a general strategy for macromolecular structure determination that includes AI-based prediction both as a starting point and as a method of model optimization.

biochemistry↗

bioRxiv2022

Endocrine disruption of early uterine differentiation causes adenocarcinoma mediated by Wnt/β-catenin- and PI3K/AKT signaling

Developmental exposure to non-mutagenic environmental factors can contribute to cancer risk, but the underlying mechanisms are not understood. We used a mouse model of endometrial adenocarcinoma that results from brief developmental exposure to an estrogenic chemical, diethylstilbestrol (DES), to determine causative factors. Single cell RNA sequencing and spatial transcriptomics of adult uteri revealed new markers of uterine epithelial stem cells, identified luminal and glandular progenitor cells, and defined a glandular epithelial differentiation trajectory. Neonatal DES exposure disrupted uterine epithelial differentiation, resulting in widespread activation of Wnt signaling and a failure to generate epithelial stem cells or distinguishable glandular and luminal epithelial cells. The endometrial stromal cells activated inflammatory signals and oxidative stress. Together, these changes activated PI3K/AKT signaling to drive malignant transformation. These findings explain how human cancers, which are often associated with abnormal activation of PI3K/AKT signaling, could result from exposure to environmental insults during development. HighlightsO_LISingle cell analysis of adult mouse uteri reveals epithelial stem cell markers and gland development trajectory C_LIO_LIDevelopmental DES exposure causes widespread activation of Wnt signaling and failure of epithelial cell differentiation C_LIO_LIUterine adenocarcinoma results from combined Wnt/{beta}-catenin activation, stromal inflammation, and induction of PI3K/AKT signaling C_LIO_LIOLFM4 is a marker of developmental DES-induced uterine adenocarcinoma C_LI

developmental biology↗

bioRxiv2022

The homeobox transcription factor DUXBL controls exit from totipotency

Upon exit from the totipotent 2-cell (2C) embryo stage, the 2C-associated transcriptional program needs to be efficiently silenced. However, the molecular mechanisms involved in this process remain mostly unknown. Here, we demonstrate that the 2C-specific transcription factor DUX directly induces the expression of DUXBL to promote this silencing. Indeed, DUX expression in Duxbl-knockout ESC causes increased induction of the 2C-transcriptional program, whereas DUXBL overexpression impairs 2C-associated transcription. CUT&RUN analyses show that DUXBL gains accessibility to DUX-bound regions in DUX-induced ESC while it is unable to bind those regions in uninduced cells. Mechanistically, we determined that DUXBL interacts with TRIM24 and TRIM33, two members of the tripartite motif superfamily involved in gene silencing and co-localizes with them in nuclear foci upon DUX expression. Furthermore, DUXBL downregulation in mouse zygotes leads to a penetrant 2C-stage arrest. Our data reveals an unexpected role for DUXBL in controlling the exit from totipotency.

developmental biology↗

bioRxiv2021

The importance of residue-level filtering, and the Top2018 best-parts dataset of high-quality protein residues

We have curated a high-quality, "best parts" reference dataset of about 3 million protein residues in about 15,000 PDB-format coordinate files, each containing only residues with good electron density support for a physically acceptable model conformation. The resulting pre-filtered data typically contains the entire core of each chain, in quite long continuous fragments. Each reference file is a single protein chain, and the total set of files were selected for low redundancy, high resolution, good MolProbity score, and other chain-level criteria. Then each residue was critically tested for adequate local map quality to firmly support its conformation, which must also be free of serious clashes or covalent-geometry outliers. The resulting Top2018 pre-filtered datasets have been released on the Zenodo online web service and is freely available for all uses under a Creative Commons license. Currently, one dataset is residue-filtered on mainchain plus C{beta} atoms, and a second dataset is full-residue filtered; each is available at 4 different sequence-identity levels. Here, we illustrate both statistics and examples that show the beneficial consequences of residue-level filtering. That process is necessary because even the best of structures contain a few highly disordered local regions with poor density and low-confidence conformations that should not be included in reference data. Therefore the open distribution of these very large, pre-filtered reference datasets constitutes a notable advance for structural bioinformatics and the fields that depend upon it. The Top2018 dataset provides the first representative sample of 3D protein structure for which excellence of experimental data constrains the detailed local conformation to be correct for essentially all 3 million residues included. Earlier generations of residue-filtered datasets were central in developing MolProbity validation used worldwide, and now Zenodo has enabled anyone to use out latest version as a sound basis for structural bioinformatics, protein design, prediction, improving biomedically important structures, or other applications.

biochemistry↗

bioRxiv2021

Superovulation does not alter calcium oscillations following fertilization

Superovulation is a common approach to maximize the number of eggs available for either clinical assisted reproductive technologies or experimental animal studies. This procedure provides supraphysiological amounts of gonadotropins to promote continued growth and maturation of ovarian follicles that otherwise would undergo atresia. There is evidence in mice, cows, sheep and humans that superovulation has a detrimental impact on the quality of the resulting ovulated eggs or embryos. Here we tested the hypothesis that eggs derived from superovulation have a reduced capacity to support calcium oscillations following fertilization, which is a critical factor in the success of embryo development. Eggs were obtained from mice that were either naturally cycling or underwent a standard superovulation protocol. Naturally cycling mice were mated to vasectomized males and vaginal plugs were checked to assure ovulation had occurred. The superovulated mice were also mated to vasectomized males for consistency of treatment across groups. The eggs were fertilized in vitro while undergoing monitoring of calcium oscillatory patterns. There were no differences in any measures of calcium oscillatory behavior, including length of the first oscillation, area under the curve of calcium signal, or frequency or persistence of oscillations. These findings indicate that superovulation does not disrupt calcium signaling at fertilization, supporting the use of this method for both clinical and experimental purposes.

developmental biology↗

bioRxiv2020

ER-Anchored Transcription Factors bZIP17 and bZIP60 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR 1 (TSAR1) and TSAR2, have been previously identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins was altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes, particularly under ER stress conditions, when they translocate from the ER to the nucleus. Furthermore, the inhibitory role of ER stress bZIP TFs in the regulation of JA-dependent terpene biosynthetic pathways appears to be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus. We postulate that activation of ER stress bZIP TFs provides the plant with a mechanism to balance metabolic activities and thereby adequately govern modulation of growth, development and defense processes in defined stress situations. One sentence summaryER stress bZIP transcription factors can interfere with the activity of jasmonate-inducible bHLH transcription factors to modulate the elicitation of plant specialized metabolism in stress conditions.

plant biology↗

bioRxiv2019

CaBLAM for CryoEM Chiropraxis, UnDowser to Rethink \"Waters\", and NGL Viewer to Recapture Online 3D Graphics in MolProbity Validation

The MolProbity web service provides macromolecular model validation to help correct local errors, for the structural biology community worldwide. Here we highlight new validation features, and also describe how we are fighting back against recent outside changes that degrade or endanger that mission. Sophisticated hacking of the MolProbity server has required continual monitoring and various security measures short of restricting user access. Refinement software now increasingly restrains validation criteria in order to supplement the sparser experimental data at 3-4[A] resolutions typical of modern cryoEM. But unfortunately the broad density allows optimization without fixing underlying problems, which means these structures often score much better than they really are. CaBLAM, our first new tool designed for this regime, was described in the previous Tools issue, and here we demonstrate its effectiveness in diagnosing local errors even when other validation outliers have been artificially removed. The deprecation of Java applets now prevents KiNG interactive online display of outliers on the 3D model during a MolProbity run, but that important functionality is now recaptured with a modified version of the Javascript NGL Viewer. Other new changes are more straightforwardly good. We are moving to the Neo4j database (graphical rather than relational), and will soon have cleaner as well as much larger reference datasets. In addition to several minor new features, we have developed a tool called UnDowser that analyzes the properties and context of modeled but clashing HOH \"waters\" to diagnose what they might actually represent. A dozen distinct scenarios are illustrated and described.

biophysics↗

bioRxiv2018

Cis-nonPro Peptides: Genuine Occurrences and their Functional Roles

While cis peptides preceding proline can occur about 5% of the time, cis peptides preceding any other residue ("cis-nonPro" peptides) are an extremely rare feature in protein structures, of considerable importance for two opposite reasons. On one hand, their genuine occurrences are mostly found at sites critical to biological function, from the active sites of carbohydrate enzymes to rare adjacent-residue disulfide bonds. On the other hand, a cis-nonPro can easily be misfit into weak or ambiguous electron density, which led to a high incidence of unjustified cis-nonPro over the 2006-2015 decade. This paper uses high-resolution crystallographic data and especially stringent quality-filtering at the residue level to identify genuine occurrences of cis-nonPro, and to survey both individual examples and broad patterns of their functionality. We explain the procedure developed to identify genuine cis-nonPro examples with almost no false positives. We then survey a large sample of the varied functional roles and structural contexts of cis-nonPro, including the uses of specific amino acids for particular purposes. We emphasize aspects not previously covered: that cis-nonPro always (except for vicinal disulfides) occur in highly ordered structure, and especially the great concentration of occurrence in proteins that process or bind carbohydrates (identified by occurrence on the CAZy website).

biochemistry↗