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.

Structural predictions of protein-DNA binding: MELD-DNA.

Structural, regulatory and enzymatic proteins interact with DNA to maintain a healthy and functional genome. Yet, our structural understanding of how proteins interact with DNA is limited. We present MELD-DNA, a novel computational approach to predict the structures of protein-DNA complexes. The method combines molecular dynamics simulations with general knowledge or experimental information through Bayesian inference. The physical model is sensitive to sequence-dependent properties and conformational changes required for binding, while information accelerates sampling of bound conformations. MELD-DNA can: (i) sample multiple binding modes; (ii) identify the preferred binding mode from the ensembles; and (iii) provide qualitative binding preferences between DNA sequences. We first assess performance on a dataset of 15 protein-DNA complexes and compare it with state-of-the-art methodologies. Furthermore, for three selected complexes, we show sequence dependence effects of binding in MELD predictions. We expect that the results presented herein, together with the freely available software, will impact structural biology (by complementing DNA structural databases) and molecular recognition (by bringing new insights into aspects governing protein-DNA interactions).

Computational insights into pH-dependence of structure and dynamics of pyrazinamidase: A comparison of wild type and mutants.

The mycobacterial enzyme pyrazinamidase (PZase) is the target of key tuberculosis drug, pyrazinamide. Mutations in PZase cause drug resistance. Herein, three point mutations, W68G, L85P, and V155G, were investigated through over 8 µs of molecular dynamics simulations coupled with essential dynamics and binding pocket analysis at neutral (pH = 7) and acidic (pH = 4) ambient conditions. The 51-71 flap region exhibited drastic displacement leading to enlargement of binding cavity, especially at the lower pH. Accessibility of solvent to the active site of the mutant enzymes was also reduced. The protonation of key surface residues at low pH results in more contribution of these residues to structural stability and integrity of the enzyme and reduced interactions with solvent molecules, which acts as a cage, keeping the enzyme together. The observed results suggest a pattern of structural alterations due to point mutations in PZase, which is consistent with other experimental and theoretical investigations and, can be harnessed for drug design purposes.

A folding motif formed with an expanded genetic alphabet.

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ-:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ-:dZ pairs join parallel strands in a four-stranded compact down-up-down-up fold. These have two possible structures: one with intercalated dZ-:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

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.

Searching for Low Probability Opening Events in a DNA Sliding Clamp.

The ß subunit of E. coli DNA polymererase III is a DNA sliding clamp associated with increasing the processivity of DNA synthesis. In its free form, it is a circular homodimer structure that can accomodate double-stranded DNA in a nonspecific manner. An open state of the clamp must be accessible before loading the DNA. The opening mechanism is still a matter of debate, as is the effect of bound DNA on opening/closing kinetics. We use a combination of atomistic, coarse-grained, and enhanced sampling strategies in both explicit and implicit solvents to identify opening events in the sliding clamp. Such simulations of large nucleic acid and their complexes are becoming available and are being driven by improvements in force fields and the creation of faster computers. Different models support alternative opening mechanisms, either through an in-plane or out-of-plane opening event. We further note some of the current limitations, despite advances, in modeling these highly charged systems with implicit solvent.

Importance of Anion-π Interactions in RNA GAAA and GGAG Tetraloops: A Combined MD and QM Study.

In this study, we demonstrate that anion-π interactions (an attractive noncovalent force between electron deficient π-systems and anions) are involved in the stabilization of GAAA and GGAG RNA tetraloops. Using the single recognition particle (SRP)-RNA complexes as a case of study, we combined molecular dynamics (MD) and quantum mechanics (QM) calculations to shed light on the structural influence of phosphate-G anion-π interactions and hydrogen bonds (HBs) involving K+/Mg2+ water clusters. In addition, the RNA assemblies herein were further characterized by means of the "atoms in molecules" (AIM) and noncovalent interactions plot (NCIplot) methodologies. We believe the results derived from this study might be important in the fields of chemical biology (RNA folding and engineering) and supramolecular chemistry (anion-π interactions) as well as to further expand the current knowledge regarding RNA structural motifs.