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We describe a computational approach to building and simulating realistic 3D models of very large RNA molecules (>1000 nucleotides) at a resolution of one "bead" per nucleotide. The method starts with a predicted secondary structure and uses several stages of energy minimization and Brownian dynamics (BD) simulation to build 3D models. A key step in the protocol is the temporary addition of a 4th spatial dimension that allows all predicted helical elements to become disentangled from each other in an effectively automated way. We then use the resulting 3D models as input to Brownian dynamics simulations that include hydrodynamic interactions (HIs) that allow the diffusive properties of the RNA to be modelled as well as enabling its conformational dynamics to be simulated. To validate the dynamics part of the method, we first show that when applied to small RNAs with known 3D structures the BD-HI simulation models accurately reproduce their experimental hydrodynamic radii (Rh). We then apply the modelling and simulation protocol to a variety of RNAs for which experimental Rh values have been reported ranging in size from 85 to 3569 nucleotides. We show that the 3D models, when used in BD-HI simulations, produce hydrodynamic radii that are usually in good agreement with experimental estimates for RNAs that do not contain tertiary contacts that persist even under very low salt conditions. Finally, we show that sampling of the conformational dynamics of large RNAs on timescales of 100 μs is computationally feasible with BD-HI simulations.

In Gram-negative bacteria, several trans-envelope complexes (TECs) have been identified that span the periplasmic space in order to facilitate lipid transport between the inner- and outer-membranes. While partial or near-complete structures of some of these TECs have been solved by conventional experimental techniques, most remain incomplete. Here we describe how a combination of computational approaches, constrained by experimental data, can be used to build complete atomic models for four TECs implicated in lipid transport in Escherichia coli. We use the DeepMind protein structure prediction algorithm, AlphaFold2, and a variant of it designed to predict protein complexes, AF2Complex, to predict the oligomeric states of key components of TECs and their likely interfaces with other components. After obtaining initial models of the complete TECs by superimposing predicted structures of subcomplexes, we use the membrane orientation prediction algorithm OPM to predict the likely orientations of the inner- and outer-membrane components in each TEC. Since, in all cases, the predicted membrane orientations in these initial models are tilted relative to each other, we devise a novel molecular mechanics-based strategy that we call membrane morphing that adjusts each TEC model until the two membranes are properly aligned with each other and separated by a distance consistent with estimates of the periplasmic width in E. coli. The study highlights the potential power of combining computational methods, operating within limits set by both experimental data and by cell physiology, for producing useable atomic structures of very large protein complexes.

The interpretation of experimental studies of co-translational protein folding often benefits from the use of computational methods that seek to model the nascent chain and its interactions with the ribosome. Ribosome-nascent chain (RNC) constructs studied experimentally can vary significantly in size and the extent to which they contain secondary and tertiary structure, and building realistic 3D models of them therefore often requires expert knowledge. To circumvent this issue, we describe here AutoRNC, an automated modeling program capable of constructing large numbers of plausible atomic models of RNCs within minutes. AutoRNC takes input from the user specifying any regions of the nascent chain that contain secondary or tertiary structure and attempts to build conformations compatible with those specifications — and with the constraints imposed by the ribosome — by sampling and progressively piecing together dipeptide conformations extracted from the RCSB. We first show that conformations of completely unfolded proteins built by AutoRNC in the absence of the ribosome have radii of gyration that match well with the corresponding experimental data. We then show that AutoRNC can build plausible conformations for a wide range of RNC constructs for which experimental data have already been reported. Since AutoRNC requires only modest computational resources, we anticipate that it will prove to be a useful hypothesis generator for experimental studies, for example, in providing indications of whether designed constructs are likely to be capable of folding, as well as providing useful starting points for downstream atomic or coarse-grained simulations of the conformational dynamics of RNCs.

DeepMind′s AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn-binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of Fe-S cluster or Zn-binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.