Outcomes of the EMDataResource cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

When will RNA get its AlphaFold moment?

The protein structure prediction problem has been solved for many types of proteins by AlphaFold. Recently, there has been considerable excitement to build off the success of AlphaFold and predict the 3D structures of RNAs. RNA prediction methods use a variety of techniques, from physics-based to machine learning approaches. We believe that there are challenges preventing the successful development of deep learning-based methods like AlphaFold for RNA in the short term. Broadly speaking, the challenges are the limited number of structures and alignments making data-hungry deep learning methods unlikely to succeed. Additionally, there are several issues with the existing structure and sequence data, as they are often of insufficient quality, highly biased and missing key information. Here, we discuss these challenges in detail and suggest some steps to remedy the situation. We believe that it is possible to create an accurate RNA structure prediction method, but it will require solving several data quality and volume issues, usage of data beyond simple sequence alignments, or the development of new less data-hungry machine learning methods.

A unified dinucleotide alphabet describing both RNA and DNA structures.

By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them observed for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical analysis of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased analysis of nucleic acid structures by structural and molecular biologists.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

Femtosecond-to-nanosecond dynamics of flavin mononucleotide monitored by stimulated Raman spectroscopy and simulations.

Flavin mononucleotide (FMN) belongs to the large family of flavins, ubiquitous yellow-coloured biological chromophores that contain an isoalloxazine ring system. As a cofactor in flavoproteins, it is found in various enzymes and photosensory receptors, like those featuring the light-oxygen-voltage (LOV) domain. The photocycle of FMN is triggered by blue light and proceeds via a cascade of intermediate states. In this work, we have studied isolated FMN in an aqueous solution in order to elucidate the intrinsic electronic and vibrational changes of the chromophore upon excitation. The ultrafast transitions of excited FMN were monitored through the joint use of femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy encompassing a time window between 0 ps and 6 ns with 50 fs time resolution. Global analysis of the obtained transient visible absorption and transient Raman spectra in combination with extensive quantum chemistry calculations identified unambiguously the singlet and triplet FMN populations and addressed solvent dynamics effects. The good agreement between the experimental and theoretical spectra facilitated the assignment of electronic transitions and vibrations. Our results represent the first steps towards more complex experiments aimed at tracking structural changes of FMN embedded in light-inducible proteins upon photoexcitation.

Interferons type II and their receptors R1 and R2 in fish species: Evolution, structure, and function.

Interferon gamma (IFN-γ) is one of the key players in the immune system of vertebrates. The evolution and properties of IFN-γ and its receptors in fish species are of special interest as they point to the origin of innate immunity in vertebrates. We studied the phylogeny, biophysical and structural properties of IFN-γ and its receptors. Our phylogeny analysis suggests the existence of two groups of IFN-γ related proteins, one specific for Acanthomorpha, the other for Cypriniformes, Characiformes and Siluriformes. The analysis further shows an ancient duplication of the gene for IFN-γ receptor 1 (IFN- γR1) and the parallel existence of the duplicated genes in all current teleost fish species. In contrast, only one gene can be found for receptor 2, IFN- γR2. The specificity of the interaction between IFN- γ and both types of IFN- γR1 was determined by microscale thermophoresis measurements of the equilibrium dissociation constants for the proteins from three fish species. The measured preference of IFN- γ for one of the two forms of receptor 1agrees with the bioinformatic analysis of the coevolution between IFN- γ and receptor 1. To elucidate structural relationships between IFN-γ of fish and other vertebrate species, we determined the crystal structure of IFN-γ from olive flounder (Paralichthys olivaceus, PoliIFN-γ) at crystallographic resolution of 2.3 Šand the low-resolution structures of Takifugu rubripes, Oreochromis niloticus, and Larimichthys crocea IFN-γ by small angle X-ray diffraction. The overall PoliIFN-γ fold is the same as the fold of the other known IFN- γ structures but there are some significant structural differences, namely the additional C-terminal helix G and a different angle between helices C and D in PoliIFN-γ.

DNATCO: assignment of DNA conformers at dnatco.org.

The web service DNATCO (dnatco.org) classifies local conformations of DNA molecules beyond their traditional sorting to A, B and Z DNA forms. DNATCO provides an interface to robust algorithms assigning conformation classes called NTC: to dinucleotides extracted from DNA-containing structures uploaded in PDB format version 3.1 or above. The assigned dinucleotide NTC: classes are further grouped into DNA structural alphabet NTA: , to the best of our knowledge the first DNA structural alphabet. The results are presented at two levels: in the form of user friendly visualization and analysis of the assignment, and in the form of a downloadable, more detailed table for further analysis offline. The website is free and open to all users and there is no login requirement.

Hydration of proteins and nucleic acids: Advances in experiment and theory. A review.

BACKGROUND: Most biological processes involve water, and the interactions of biomolecules with water affect their structure, function and dynamics. SCOPE OF REVIEW: This review summarizes the current knowledge of protein and nucleic acid interactions with water, with a special focus on the biomolecular hydration layer. Recent developments in both experimental and computational methods that can be applied to the study of hydration structure and dynamics are reviewed, including software tools for the prediction and characterization of hydration layer properties. MAJOR CONCLUSIONS: In the last decade, important advances have been made in our understanding of the factors that determine how biomolecules and their aqueous environment influence each other. Both experimental and computational methods contributed to the gradually emerging consensus picture of biomolecular hydration. GENERAL SIGNIFICANCE: An improved knowledge of the structural and thermodynamic properties of the hydration layer will enable a detailed understanding of the various biological processes in which it is involved, with implications for a wide range of applications, including protein-structure prediction and structure-based drug design.

WatAA: Atlas of Protein Hydration. Exploring synergies between data mining and ab initio calculations.

Water molecules represent an integral part of proteins and a key determinant of protein structure, dynamics and function. WatAA is a newly developed, web-based atlas of amino-acid hydration in proteins. The atlas provides information about the ordered first hydration shell of the most populated amino-acid conformers in proteins. The data presented in the atlas are drawn from two sources: experimental data and ab initio quantum-mechanics calculations. The experimental part is based on a data-mining study of a large set of high-resolution protein crystal structures. The crystal-derived data include 3D maps of water distribution around amino-acids and probability of occurrence of each of the identified hydration sites. The quantum mechanics calculations validate and extend this primary description by optimizing the water position for each hydration site, by providing hydrogen atom positions and by quantifying the interaction energy that stabilizes the water molecule at the particular hydration site position. The calculations show that the majority of experimentally derived hydration sites are positioned near local energy minima for water, and the calculated interaction energies help to assess the preference of water for the individual hydration sites. We propose that the atlas can be used to validate water placement in electron density maps in crystallographic refinement, to locate water molecules mediating protein-ligand interactions in drug design, and to prepare and evaluate molecular dynamics simulations. WatAA: Atlas of Protein Hydration is freely available without login at .

Bioinformatic analysis of the protein/DNA interface.

To investigate the principles driving recognition between proteins and DNA, we analyzed more than thousand crystal structures of protein/DNA complexes. We classified protein and DNA conformations by structural alphabets, protein blocks [de Brevern, Etchebest and Hazout (2000) (Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks. Prots. Struct. Funct. Genet., 41:271-287)] and dinucleotide conformers [Svozil, Kalina, Omelka and Schneider (2008) (DNA conformations and their sequence preferences. Nucleic Acids Res., 36:3690-3706)], respectively. Assembling the mutually interacting protein blocks and dinucleotide conformers into 'interaction matrices' revealed their correlations and conformer preferences at the interface relative to their occurrence outside the interface. The analyzed data demonstrated important differences between complexes of various types of proteins such as transcription factors and nucleases, distinct interaction patterns for the DNA minor groove relative to the major groove and phosphate and importance of water-mediated contacts. Water molecules mediate proportionally the largest number of contacts in the minor groove and form the largest proportion of contacts in complexes of transcription factors. The generally known induction of A-DNA forms by complexation was more accurately attributed to A-like and intermediate A/B conformers rare in naked DNA molecules.

Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures.

Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon-donor hydrogen bonds, OH-π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.

Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes.

Monitoring RAYT activity by surface plasmon resonance biosensor.

The process of DNA transposition involves the binding, cleavage, and recombination of specific DNA segments (transposable elements, TE) and is catalyzed by special enzymes encoded by the TE transposases. REP-associated tyrosine transposases (RAYTs) are a class of Y1 nucleases related to the IS200/IS605 transposases associated with a bacterial TE known as repetitive extragenic palindrome elements (REPs). Although RAYT has been subject of numerous studies, where DNA binding and cleavage by RAYT have been confirmed for Escherichia coli, the molecular mechanism of DNA insertion has not been fully understood. In this work, it is demonstrated that surface plasmon resonance (SPR) biosensor technology combined with a system of DNA hairpin probes (mimicking the natural REP sequence) and short oligonucleotides (ONs) can provide a rapid and real-time platform for monitoring and quantification of RAYT activity. We utilized RAYT from E. coli (strain MG1655) as a model system, where we evaluated its activity towards both a natural REP sequence as well as REP sequences having modifications targeting specific features of the DNA crucial for the DNA binding and cleavage. The characteristics of the RAYT-DNA interaction obtained by means of the SPR approach were compared with the results of SDS-PAGE analysis.

Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

Local dynamics of proteins and DNA evaluated from crystallographic B factors.

The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein-DNA crystal structures were analyzed [Schneider et al. (2014), Nucleic Acids Res. 42, 3381-3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein-protein or protein-DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are surprising. The features discriminating different types of residues are less pronounced in structures with lower crystallographic resolution. Some of the observed trends are likely to be the consequence of improper refinement protocols that may need to be rectified.

Outcomes of the EMDataResource Cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein/nucleic-acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: E. coli beta-galactosidase with inhibitor, SARS-CoV-2 RNA-dependent RNA polymerase with covalently bound nucleotide analog, and SARS-CoV-2 ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. We found that (1) the quality of submitted ligand models and surrounding atoms varied, as judged by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics, and contact scores, and (2) a composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

Automatic workflow for the classification of local DNA conformations.

BACKGROUND: A growing number of crystal and NMR structures reveals a considerable structural polymorphism of DNA architecture going well beyond the usual image of a double helical molecule. DNA is highly variable with dinucleotide steps exhibiting a substantial flexibility in a sequence-dependent manner. An analysis of the conformational space of the DNA backbone and the enhancement of our understanding of the conformational dependencies in DNA are therefore important for full comprehension of DNA structural polymorphism. RESULTS: A detailed classification of local DNA conformations based on the technique of Fourier averaging was published in our previous work. However, this procedure requires a considerable amount of manual work. To overcome this limitation we developed an automatic classification method consisting of the combination of supervised and unsupervised approaches. A proposed workflow is composed of k-NN method followed by a non-hierarchical single-pass clustering algorithm. We applied this workflow to analyze 816 X-ray and 664 NMR DNA structures released till February 2013. We identified and annotated six new conformers, and we assigned four of these conformers to two structurally important DNA families: guanine quadruplexes and Holliday (four-way) junctions. We also compared populations of the assigned conformers in the dataset of X-ray and NMR structures. CONCLUSIONS: In the present work we developed a machine learning workflow for the automatic classification of dinucleotide conformations. Dinucleotides with unassigned conformations can be either classified into one of already known 24 classes or they can be flagged as unclassifiable. The proposed machine learning workflow permits identification of new classes among so far unclassifiable data, and we identified and annotated six new conformations in the X-ray structures released since our previous analysis. The results illustrate the utility of machine learning approaches in the classification of local DNA conformations.

Evolution of REP diversity: a comparative study.

BACKGROUND: Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution. RESULTS: Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts. CONCLUSIONS: The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells.

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light.

Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.

Diffraction anisotropy and paired refinement: crystal structure of H33, a protein binder to interleukin 10.

Binder H33 is a small protein binder engineered by ribosome display to bind human interleukin 10. Crystals of binder H33 display severe diffraction anisotropy. A set of data files with correction for diffraction anisotropy based on different local signal-to-noise ratios was prepared. Paired refinement was used to find the optimal anisotropic high-resolution diffraction limit of the data: 3.13-2.47 Å. The structure of binder H33 belongs to the 2% of crystal structures with the highest solvent content in the Protein Data Bank.