Adaptive Ensemble Refinement of Protein Structures in High Resolution Electron Microscopy Density Maps with Radical Augmented Molecular Dynamics Flexible Fitting.

Recent advances in cryo-electron microscopy (cryo-EM) have enabled modeling macromolecular complexes that are essential components of the cellular machinery. The density maps derived from cryo-EM experiments are often integrated with manual, knowledge-driven or artificial intelligence-driven and physics-guided computational methods to build, fit, and refine molecular structures. Going beyond a single stationary-structure determination scheme, it is becoming more common to interpret the experimental data with an ensemble of models that contributes to an average observation. Hence, there is a need to decide on the quality of an ensemble of protein structures on-the-fly while refining them against the density maps. We introduce such an adaptive decision-making scheme during the molecular dynamics flexible fitting (MDFF) of biomolecules. Using RADICAL-Cybertools, the new RADICAL augmented MDFF implementation (R-MDFF) is examined in high-performance computing environments for refinement of two prototypical protein systems, adenylate kinase and carbon monoxide dehydrogenase. For these test cases, use of multiple replicas in flexible fitting with adaptive decision making in R-MDFF improves the overall correlation to the density by 40% relative to the refinements of the brute-force MDFF. The improvements are particularly significant at high, 2-3 Å map resolutions. More importantly, the ensemble model captures key features of biologically relevant molecular dynamics that are inaccessible to a single-model interpretation. Finally, the pipeline is applicable to systems of growing sizes, which is demonstrated using ensemble refinement of capsid proteins from the chimpanzee adenovirus. The overhead for decision making remains low and robust to computing environments. The software is publicly available on GitHub and includes a short user guide to install R-MDFF on different computing environments, from local Linux-based workstations to high-performance computing environments.

Exploring cryo-electron microscopy with molecular dynamics.

Single particle analysis cryo-electron microscopy (EM) and molecular dynamics (MD) have been complimentary methods since cryo-EM was first applied to the field of structural biology. The relationship started by biasing structural models to fit low-resolution cryo-EM maps of large macromolecular complexes not amenable to crystallization. The connection between cryo-EM and MD evolved as cryo-EM maps improved in resolution, allowing advanced sampling algorithms to simultaneously refine backbone and sidechains. Moving beyond a single static snapshot, modern inferencing approaches integrate cryo-EM and MD to generate structural ensembles from cryo-EM map data or directly from the particle images themselves. We summarize the recent history of MD innovations in the area of cryo-EM modeling. The merits for the myriad of MD based cryo-EM modeling methods are discussed, as well as, the discoveries that were made possible by the integration of molecular modeling with cryo-EM. Lastly, current challenges and potential opportunities are reviewed.

Poor Person's pH Simulation of Membrane Proteins.

pH conditions are central to the functioning of all biomolecules. However, implications of pH changes are nontrivial on a molecular scale. Though a rigorous microscopic definition of pH exists, its implementation in classical molecular dynamics (MD) simulations is cumbersome, and more so in large integral membrane systems. In this chapter, an integrative pipeline is described that combines Multi-Conformation Continuum Electrostatics (MCCE) computations with MD simulations to capture the effect of transient protonation states on the coupled conformational changes in transmembrane proteins. The core methodologies are explained, and all the software required to set up this pipeline are outlined with their key parameters. All associated analyses of structure and function are provided using two case studies, namely those of bioenergetic complexes: NADH dehydrogenase (complex I) and Vo domain of V-type ATPase. The hybrid MCCE-MD pipeline has allowed the discovery of hydrogen bond networks, ligand binding pathways, and disease-causing mutations.

Data-guided Multi-Map variables for ensemble refinement of molecular movies.

Driving molecular dynamics simulations with data-guided collective variables offer a promising strategy to recover thermodynamic information from structure-centric experiments. Here, the three-dimensional electron density of a protein, as it would be determined by cryo-EM or x-ray crystallography, is used to achieve simultaneously free-energy costs of conformational transitions and refined atomic structures. Unlike previous density-driven molecular dynamics methodologies that determine only the best map-model fits, our work employs the recently developed Multi-Map methodology to monitor concerted movements within equilibrium, non-equilibrium, and enhanced sampling simulations. Construction of all-atom ensembles along the chosen values of the Multi-Map variable enables simultaneous estimation of average properties, as well as real-space refinement of the structures contributing to such averages. Using three proteins of increasing size, we demonstrate that biased simulation along the reaction coordinates derived from electron densities can capture conformational transitions between known intermediates. The simulated pathways appear reversible with minimal hysteresis and require only low-resolution density information to guide the transition. The induced transitions also produce estimates for free energy differences that can be directly compared to experimental observables and population distributions. The refined model quality is superior compared to those found in the Protein Data Bank. We find that the best quantitative agreement with experimental free-energy differences is obtained using medium resolution density information coupled to comparatively large structural transitions. Practical considerations for probing the transitions between multiple intermediate density states are also discussed.

Molecular Dynamics Flexible Fitting: All You Want to Know About Resolution Exchange.

In recent years, owing to the advances in instrumentation, cryo-EM has emerged as the go-to tool for obtaining high-resolution structures of biomolecular systems. However, building three-dimensional atomic structures of biomolecules from these high-resolution maps remains a concern for the traditional map-guided structure-determination schemes. Recently, we developed a computational tool, Resolution Exchange Molecular Dynamics Flexible Fitting (ReMDFF) to address this problem by re-refining a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution (Wang et al., J Struct Biol 204(2):319-328, 2018). In this chapter, we present a step-by-step outline for preparing, executing, and analyzing ReMDFF refinements of simple proteins and multimeric complexes. The structure determination of carbon monoxide dehydrogenase and Mg2+-channel CorA are employed as case studies. All scripts are provided via GitHub (Vant, Resolution exchange molecular dynamics flexible fitting (ReMDFF) all you want to know about flexible fitting, 2019, https://github.com/jvant/ReMDFF_Singharoy_Group.git ).

Flexible Fitting of Small Molecules into Electron Microscopy Maps Using Molecular Dynamics Simulations with Neural Network Potentials.

Despite significant advances in resolution, the potential for cryo-electron microscopy (EM) to be used in determining the structures of protein-drug complexes remains unrealized. Determination of accurate structures and coordination of bound ligands necessitates simultaneous fitting of the models into the density envelopes, exhaustive sampling of the ligand geometries, and, most importantly, concomitant rearrangements in the side chains to optimize the binding energy changes. In this article, we present a flexible-fitting pipeline where molecular dynamics flexible fitting (MDFF) is used to refine structures of protein-ligand complexes from 3 to 5 Å electron density data. Enhanced sampling is employed to explore the binding pocket rearrangements. To provide a model that can accurately describe the conformational dynamics of the chemically diverse set of small-molecule drugs inside MDFF, we use QM/MM and neural-network potential (NNP)/MM models of protein-ligand complexes, where the ligand is represented using the QM or NNP model, and the protein is represented using established molecular mechanical force fields (e.g., CHARMM). This pipeline offers structures commensurate to or better than recently submitted high-resolution cryo-EM or X-ray models, even when given medium to low-resolution data as input. The use of the NNPs makes the algorithm more robust to the choice of search models, offering a radius of convergence of 6.5 Å for ligand structure determination. The quality of the predicted structures was also judged by density functional theory calculations of ligand strain energy. This strain potential energy is found to systematically decrease with better fitting to density and improved ligand coordination, indicating correct binding interactions. A computationally inexpensive protocol for computing strain energy is reported as part of the model analysis protocol that monitors both the ligand fit as well as model quality.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.