The RNA genome of measles virus is encapsidated by the nucleoprotein within a helical nucleocapsid that serves as a template for both transcription and replication. The intrinsically disordered domain of the nucleoprotein (NTAIL) is essential for binding the polymerase complex responsible for viral transcription and replication. As for many IDPs, binding of NTAIL occurs through a short molecular recognition element (MoRE) that folds upon binding, with the majority of NTAIL remaining disordered. Although NTAIL regions far from the MoRE influence the binding affinity, interactions between them and the MoRE have not been investigated in depth. Relying on photo-induced electron transfer (PET) experiments between tryptophan and cysteine pairs placed at different positions in the protein under varying salt and pH conditions, combined with analytical models, simulations, and coevolutionary analysis, we identified transient interactions between two disordered regions distant in sequence, which dominate NTAIL dynamics, and regulate the conformational preferences of both the MoRE and the entire NTAIL domain. We propose mechanisms by which these non-local interactions may regulate binding to the measles phosphoprotein, polymerase recruitment, and ultimately viral transcription and replication. Our findings may be extended to other IDPs, where non-local intra-protein interactions affect the conformational preferences of intermolecular binding sites. The measles virus relies on the intrinsically disordered domain of its nucleoprotein, NTAIL, to bind the polymerase complex responsible for viral transcription and replication, but the role played by disordered regions away from the binding site is not clearly understood. Here, through a combination of experiments and simulations, the authors show that transient and non-local interactions between disordered regions distant in sequence influence the conformational preferences of the binding sites and the folding and availability of its molecular recognition element, affecting viral replication kinetics.

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.

Photoinduced electron transfer (PET) relaxation is a fluorescence spectroscopy method that probes the contact dynamics between a fluorophore and a quencher, e.g. tryptophan (W) and cysteine (C). PET measurements can provide valuable information on the intrachain dynamics of intrinsically disordered proteins (IDPs). Simple homopolymer models provide a useful guide for interpreting PET measurements, as they can estimate PET quenching times for W-C pairs flanking a fully disordered sequence as a function of sequence separation.

Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation. Using atomic force microscopy, Pan et al. show that cyclic nucleotide-gated ion channel SthK, which can be differentially activated by cAMP and cGMP, binds both cyclic nucleotides but only cAMP can access a deep-bound state that could be essential for cAMP-dependent channel activation.

The Measles virus nucleocapsid is made of thousands of nucleoprotein (N) repeats, which hold the viral RNA in a helical structure. The last 125 amino acids of each N repeat (NTAIL) are intrinsically disordered and protrude radially outward from the nucleocapsid. NTAIL promotes virus replication by binding to the XD domain of the phosphoprotein P (PXD), which in turn brings the viral polymerase close to the nucleocapsid, where it transcribes and replicates the viral RNA. Only 18 amino acids of NTAIL directly bind to PXD via coupled folding and binding. The majority of NTAIL, on either side of this molecular recognition region (MoRE), remains disordered. While it has been shown that these disordered regions dampen the binding affinity, interactions involving these regions, and their possible functional role have not been identified.

Cyclic nucleotide (cN) gated ion channels such as the SthK channel are crucial in many physiological processes. Strangely, and despite their chemical similarity, cAMP acts as an activator while cGMP as an inhibitor of the SthK channel. We combined atomic force microscopy (AFM) and molecular dynamics (MD) simulations to investigate the mechanism of cyclic nucleotide binding domain (CNBD) discrimination between cAMP and cGMP. While short contact time AFM measurements revealed similar binding strengths of cAMP and cGMP, from longer contact times results suggest that ligand detection originates from the difference in binding modes of cAMP and cGMP.

NTAIL is the intrinsically disordered C-terminal domain of the measles virus nucleoprotein N. NTAIL protrudes from the nucleocapsid where the viral RNA is packaged. It recruits the polymerase and enables viral replication upon binding to a folded domain of protein P (PXD). The binding occurs via a coupled folding and binding mechanism, in which a small NTAIL region acts as binding recognition motive and folds into an κ-helix (κ-MoRE). The remaining portion of NTAIL remains disordered upon binding.

Modern cryo-EM provides high-resolution structures of multiple conformational states of biomolecules, yielding insights into their conformational heterogeneity and functional mechanisms. Molecular dynamics simulations can yield transition pathways and free energies along these paths. Unfortunately, due to their complexity, most systems cannot be fully sampled with current computational resources, such that one has to resort to enhanced sampling along a suitable reaction coordinate. Here, the choice of such a reaction coordinate is crucial but non-trivial.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

The measles virus nucleoprotein N possesses an intrinsically disordered domain, NTAIL, that protrudes from the nucleocapsid and binds to the P protein, thereby recruiting the polymerase and enabling viral replication. A small NTAIL region (18 out of 125 aa) acts as a binding motive (α-MoRE), folding upon binding to the folded X domain of P (PXD). The rest of NTAIL remains disordered. Although the remaining disordered regions of NTAIL have been shown to dampen the binding affinity, the underlying mechanism and their role in NTAIL dynamical reconfiguration upon binding is not clear. We studied the conformational dynamics of full-length NTAIL, in the free and PXD-bound states, using photo-induced electron transfer (PET) between a single tryptophan and a cysteine, placed at different labelling positions, spanning different regions of the protein. We find that full-length NTAIL exhibits significant dynamical heterogeneity, as measured by contact formation rates. These rates can be directly compared to rates generated from polymer models, coarse-grained and all-atom simulations. By comparing PET and CD measurements of full-length NTAIL and α-MoRE peptides under various conditions (e.g. pH, salt, and helix inducing solvents), with analytical polymer models, and with coarse-grained simulations, we find that neither local properties of the α-MoRE (secondary structure sampling), nor charge interactions alone can quantitatively explain our experimental observations. All atom simulations of full-length NTAIL, and of the fragment free in solution, suggest a significant slowing down of contact formation dynamics due to non-local interactions involving the flanking disordered regions. We discuss the role of charge, local secondary structure interactions, and non-local interactions, in the observed dynamical slowing down in the α-MoRE region of NTAIL. Our results highlight the potential role of flanking regions in the function and binding of NTAIL to PXD.

Modern cryo-EM provides high-resolution structures of multiple conformational states of biomolecules and biomolecular complexes from heterogeneous samples, yielding spectacular insights into their conformational heterogeneity and functional mechanisms. This approach is limited, however, to highly populated states and lacks a detailed description of the dynamics and energetics of the transitions between these states. Here we show that molecular dynamics (MD) -based structure refinement methods can be used not only to obtain such information, but also to provide time resolved pathways for each atom of the system. As a proof of concept, we use the correlation driven MD (CDMD) structure refinement method to investigate the conformational transition between open and closed states of a ligand free Adenylate-kinase (AKE) protein in solution. In CDMD the simulated system is driven from its initial state to the target state by introducing, in addition to the MD forcefield, a controlled biasing potential. This potential tends to maximize the real space correlation coefficient (CC) between the density of the simulated system and of the target state. The value of this correlation coefficient CC(X) specified for any given configuration X along the simulation trajectory can be used as a coordinate to describe the transition towards the target state. This results in different coordinates describing the forward and reverse transitions. We overcome this problem by optimizing the difference between the correlations to the start and end states, respectively. Using this reaction coordinate allows to compute the free energy profile by umbrella sampling simulations. Compared to reference free energies derived from 250 microseconds of unbiased MD simulations, our method yields similar transition pathways, thus underscoring that indeed low energy paths are identified. Our method should also be useful for larger systems for which free sampling is impossible.

We present a correlation-driven molecular dynamics (CDMD) method for automated refinement of atomistic models into cryo-electron microscopy (cryo-EM) maps at resolutions ranging from near-atomic to subnanometer. It utilizes a chemically accurate force field and thermodynamic sampling to improve the real-space correlation between the modeled structure and the cryo-EM map. Our framework employs a gradual increase in resolution and map-model agreement as well as simulated annealing, and allows fully automated refinement without manual intervention or any additional rotamer- and backbone-specific restraints. Using multiple challenging systems covering a wide range of map resolutions, system sizes, starting model geometries and distances from the target state, we assess the quality of generated models in terms of both model accuracy and potential of overfitting. To provide an objective comparison, we apply several well-established methods across all examples and demonstrate that CDMD performs best in most cases.

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource ( www.emdataresource.org ). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers and users, and (3) compare the performance of current metrics used for evaluation of models. The focus was on near-atomic resolution maps with an innovative twist: three of four target maps formed a resolution series (1.8 to 3.1 Å) from the same specimen and imaging experiment. Tools developed in previous challenges were expanded for managing, visualizing and analyzing the 63 submitted coordinate models, and several novel metrics were introduced. The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual laboratory experiments and holdings of structure data archives such as the Protein Data Bank. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived from these benchmark maps by 13 participating teams, representing both widely used and novel modeling approaches. We also evaluate the pros and cons of the commonly used metrics to assess model quality and recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed density in the cryo-EM map.

Modern cryo-electron microscopy (cryo-EM) can resolve large biomolecular complexes in different functional states, producing near-atomic resolution density maps previously attainable only by X-ray crystallography. However, advances in refinement of atomistic models into maps lag behind advances in generation of high resolution density maps. Here, we present a method for fully automated refinement of atomic models into high-resolution cryo-EM maps. The method involves correlation-based molecular dynamics fitting in real space via a continuous series of simulated maps of increasing resolution and a final step of simulated annealing. An efficient, parallel version of the correlation-based fitting algorithm is implemented within the GROMACS simulation package. Refinements of large molecular complexes can be performed in parallel with no manual intervention and no ad hoc restraints. We test our method on available structures of a rabbit muscle aldolase, two 20S proteasome complexes, and a bacterial 70S ribosome complex. Resolutions of the test systems are between 2.5Å and 3.4Å. We demonstrate that the method yields higher quality models than those produced by combining automated de novo chain building and reciprocal space refinement tools commonly used in crystallography and cryo-EM. Cross-validation against independent cryo-EM reconstructions confirms that the improved quality is not due to overfitting. Full refinement of a system requires setting up only one molecular dynamics simulation in GROMACS. The current implementation is optimized to run on medium-sized GPU clusters typically used for cryo-EM image processing. This method will contribute to drastically reducing the time and effort currently needed to produce accurate atomic models from high resolution data.

Ribosomal stalling during protein synthesis in bacteria occurs in different ways and under different conditions. Stalling of specific peptide sequences can be a pre-programmed means of detecting the presence of potentially lethal antibiotics and constitute the initial step of a complex resistance pathway. An example of this is the stalling of ErmBL peptide synthesis in the presence of the antibiotic erythromycin. In other cases, stalling seems to be an effect of unusually slow, sequence dependent, rates of amino acid incorporation. This is the case for translation of proteins containing poly-proline stretches. Poly-proline sequences are known to stall ribosomes, normal translation rates are achieved only by recruiting a special elongation factor (EF-P in bacteria). Here, we investigate the stalling mechanisms in the two scenarios described above by explicit-solvent, all-atom molecular dynamics simulations of the ribosome. The simulations are started from high-resolution cryo-EM structures and performed under stalling and non-stalling conditions. We find networks of allosteric interactions between the nascent peptide chain and the ribosome that differently affect the positioning and the dynamics of the peptidyl tRNA relative to the A-site tRNA in such a way as to hinder peptide bond formation depending on the presence of the antibiotic (in the first scenario) or the absence of the elongation factor (second scenario). The simulation results not only explain the stalling mechanism, but can also predict the effect of mutations on stalling. In the case of erythromycin induced stalling, these predictions have been experimentally confirmed by a toe-printing assay. Our results illustrate the fine details of how the efficiency of peptide bond formation can be modulated by external factors in a way that depends on the specific sequence being translated.