Dynamics of chromosome organization in a minimal bacterial cell.

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculate in silico chromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM.

Molecular dynamics simulation of an entire cell.

The ultimate microscope, directed at a cell, would reveal the dynamics of all the cell's components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on the brink of meeting this challenge. In this perspective, we show how an integrative approach can be employed to model an entire cell, the minimal cell, JCVI-syn3A, at full complexity. This step opens the way to interrogate the cell's spatio-temporal evolution with molecular dynamics simulations, an approach that can be extended to other cell types in the near future.

Toward the Complete Functional Characterization of a Minimal Bacterial Proteome.

Recently, we presented a whole-cell kinetic model of the genetically minimal bacterium JCVI-syn3A that described the coupled metabolic and genetic information processes and predicted behaviors emerging from the interactions among these networks. JCVI-syn3A is a genetically reduced bacterial cell that has the fewest number and smallest fraction of genes of unclear function, with approximately 90 of its 452 protein-coding genes (that is less than 20%) unannotated. Further characterization of unclear JCVI-syn3A genes strengthens the robustness and predictive power of cell modeling efforts and can lead to a deeper understanding of biophysical processes and pathways at the cell scale. Here, we apply computational analyses to elucidate the functions of the products of several essential but previously uncharacterized genes involved in integral cellular processes, particularly those directly affecting cell growth, division, and morphology. We also suggest directed wet-lab experiments informed by our analyses to further understand these "missing puzzle pieces" that are an essential part of the mosaic of biological interactions present in JCVI-syn3A. Our workflow leverages evolutionary sequence analysis, protein structure prediction, interactomics, and genome architecture to determine upgraded annotations. Additionally, we apply the structure prediction analysis component of our work to all 452 protein coding genes in JCVI-syn3A to expedite future functional annotation studies as well as the inverse mapping of the cell state to more physical models requiring all-atom or coarse-grained representations for all JCVI-syn3A proteins.

Metabolite Damage and Damage Control in a Minimal Genome.

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself.

Integrating cellular and molecular structures and dynamics into whole-cell models.

A complete description of the state of the cell requires knowledge of its size, shape, components, intracellular reactions, and interactions with its environment-all of these as a function of time and cell growth. Adding to this list is the need for theoretical models and simulations that integrate and help to interpret this daunting amount of experimental data. It seems like an overwhelming list of requirements, but progress is being made on many fronts. In this review, we discuss the current challenges and problems in obtaining sufficient information about each aspect of a dynamical whole-cell model (DWCM) for simple and well-studied bacterial systems.

Fundamental behaviors emerge from simulations of a living minimal cell.

We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase.

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps.

JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli's, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome's configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.

Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA.

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.

Theoretical Description of the Primary Proton-Coupled Electron Transfer Reaction in the Cytochrome bc1 Complex.

The cytochrome bc1 complex is a transmembrane enzymatic protein complex that plays a central role in cellular energy production and is present in both photosynthetic and respiratory chain organelles. Its reaction mechanism is initiated by the binding of a quinol molecule to an active site, followed by a series of charge transfer reactions between the quinol and protein subunits. Previous work hypothesized that the primary reaction was a concerted proton-coupled electron transfer (PCET) reaction because of the apparent absence of intermediate states associated with single proton or electron transfer reactions. In the present study, the kinetics of the primary bc1 complex PCET reaction is investigated with a vibronically nonadiabatic PCET theory in conjunction with all-atom molecular dynamics simulations and electronic structure calculations. The computed rate constants and relatively high kinetic isotope effects are consistent with experimental measurements on related biomimetic systems. The analysis implicates a concerted PCET mechanism with significant hydrogen tunneling and nonadiabatic effects in the bc1 complex. Moreover, the employed theoretical framework is shown to serve as a general strategy for describing PCET reactions in bioenergetic systems.

Generalized correlation-based dynamical network analysis: a new high-performance approach for identifying allosteric communications in molecular dynamics trajectories.

Molecular interactions are essential for regulation of cellular processes from the formation of multi-protein complexes to the allosteric activation of enzymes. Identifying the essential residues and molecular features that regulate such interactions is paramount for understanding the biochemical process in question, allowing for suppression of a reaction through drug interventions or optimization of a chemical process using bioengineered molecules. In order to identify important residues and information pathways within molecular complexes, the dynamical network analysis method was developed and has since been broadly applied in the literature. However, in the dawn of exascale computing, this method is frequently limited to relatively small biomolecular systems. In this work, we provide an evolution of the method, application, and interface. All data processing and analysis are conducted through Jupyter notebooks, providing automatic detection of important solvent and ion residues, an optimized and parallel generalized correlation implementation that is linear with respect to the number of nodes in the system, and subsequent community clustering, calculation of betweenness of contacts, and determination of optimal paths. Using the popular visualization program visual molecular dynamics (VMD), high-quality renderings of the networks over the biomolecular structures can be produced. Our new implementation was employed to investigate three different systems, with up to 2.5M atoms, namely, the OMP-decarboxylase, the leucyl-tRNA synthetase complexed with its cognate tRNA and adenylate, and respiratory complex I in a membrane environment. Our enhanced and updated protocol provides the community with an intuitive and interactive interface, which can be easily applied to large macromolecular complexes.

Scalable molecular dynamics on CPU and GPU architectures with NAMD.

NAMDis a molecular dynamics program designed for high-performance simulations of very large biological objects on CPU- and GPU-based architectures. NAMD offers scalable performance on petascale parallel supercomputers consisting of hundreds of thousands of cores, as well as on inexpensive commodity clusters commonly found in academic environments. It is written in C++ and leans on Charm++ parallel objects for optimal performance on low-latency architectures. NAMD is a versatile, multipurpose code that gathers state-of-the-art algorithms to carry out simulations in apt thermodynamic ensembles, using the widely popular CHARMM, AMBER, OPLS, and GROMOS biomolecular force fields. Here, we review the main features of NAMD that allow both equilibrium and enhanced-sampling molecular dynamics simulations with numerical efficiency. We describe the underlying concepts utilized by NAMD and their implementation, most notably for handling long-range electrostatics; controlling the temperature, pressure, and pH; applying external potentials on tailored grids; leveraging massively parallel resources in multiple-copy simulations; and hybrid quantum-mechanical/molecular-mechanical descriptions. We detail the variety of options offered by NAMD for enhanced-sampling simulations aimed at determining free-energy differences of either alchemical or geometrical transformations and outline their applicability to specific problems. Last, we discuss the roadmap for the development of NAMD and our current efforts toward achieving optimal performance on GPU-based architectures, for pushing back the limitations that have prevented biologically realistic billion-atom objects to be fruitfully simulated, and for making large-scale simulations less expensive and easier to set up, run, and analyze. NAMD is distributed free of charge with its source code at www.ks.uiuc.edu.

An in-silico human cell model reveals the influence of spatial organization on RNA splicing.

Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.

Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations.

To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

Multiscale modeling and cinematic visualization of photosynthetic energy conversion processes from electronic to cell scales.

Conversion of sunlight into chemical energy, namely photosynthesis, is the primary energy source of life on Earth. A visualization depicting this process, based on multiscale computational models from electronic to cell scales, is presented in the form of an excerpt from the fulldome show Birth of Planet Earth. This accessible visual narrative shows a lay audience, including children, how the energy of sunlight is captured, converted, and stored through a chain of proteins to power living cells. The visualization is the result of a multi-year collaboration among biophysicists, visualization scientists, and artists, which, in turn, is based on a decade-long experimental-computational collaboration on structural and functional modeling that produced an atomic detail description of a bacterial bioenergetic organelle, the chromatophore. Software advancements necessitated by this project have led to significant performance and feature advances, including hardware-accelerated cinematic ray tracing and instanced visualizations for efficient cell-scale modeling. The energy conversion steps depicted feature an integration of function from electronic to cell levels, spanning nearly 12 orders of magnitude in time scales. This atomic detail description uniquely enables a modern retelling of one of humanity's earliest stories-the interplay between light and life.

Stochastic Analysis Demonstrates the Dual Role of Hfq in Chaperoning E. coli Sugar Shock Response.

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or "sugar shock" in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

Kinetic Modeling of the Genetic Information Processes in a Minimal Cell.

JCVI-syn3A is a minimal bacterial cell with a 543 kbp genome consisting of 493 genes. For this slow growing minimal cell with a 105 min doubling time, we recently established the essential metabolism including the transport of required nutrients from the environment, the gene map, and genome-wide proteomics. Of the 452 protein-coding genes, 143 are assigned to metabolism and 212 are assigned to genetic information processing. Using genome-wide proteomics and experimentally measured kinetic parameters from the literature we present here kinetic models for the genetic information processes of DNA replication, replication initiation, transcription, and translation which are solved stochastically and averaged over 1,000 replicates/cells. The model predicts the time required for replication initiation and DNA replication to be 8 and 50 min on average respectively and the number of proteins and ribosomal components to be approximately doubled in a cell cycle. The model of genetic information processing when combined with the essential metabolic and cell growth networks will provide a powerful platform for studying the fundamental principles of life.

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.