Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells.

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria which encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semi-permeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We use molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

Electron transfer in a crystalline cytochrome with four hemes.

Diffusion of electrons over distances on the order of 100 µm has been observed in crystals of a small tetraheme cytochrome (STC) from Shewanella oneidensis [J. Huang et al. J. Am. Chem. Soc. 142, 10459-10467 (2020)]. Electron transfer between hemes in adjacent subunits of the crystal is slower and more strongly dependent on temperature than had been expected based on semiclassical electron-transfer theory. We here explore explanations for these findings by molecular-dynamics simulations of crystalline and monomeric STC. New procedures are developed for including time-dependent quantum mechanical energy differences in the gap between the energies of the reactant and product states and for evaluating fluctuations of the electronic-interaction matrix element that couples the two hemes. Rate constants for electron transfer are calculated from the time- and temperature-dependent energy gaps, coupling factors, and Franck-Condon-weighted densities of states using an expression with no freely adjustable parameters. Back reactions are considered, as are the effects of various protonation states of the carboxyl groups on the heme side chains. Interactions with water are found to dominate the fluctuations of the energy gap between the reactant and product states. The calculated rate constant for electron transfer from heme IV to heme Ib in a neighboring subunit at 300 K agrees well with the measured value. However, the calculated activation energy of the reaction in the crystal is considerably smaller than observed. We suggest two possible explanations for this discrepancy. The calculated rate constant for transfer from heme I to II within the same subunit of the crystal is about one-third that for monomeric STC in solution.

The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees.

PLAT domain protein 1 (PLAT1/PLAFP) binds to the Arabidopsis thaliana plasma membrane and inserts a lipid.

Robust agricultural yields depend on the plant's ability to fix carbon amid variable environmental conditions. Over seasonal and diurnal cycles, the plant must constantly adjust its metabolism according to available resources or external stressors. The metabolic changes that a plant undergoes in response to stress are well understood, but the long-distance signaling mechanisms that facilitate communication throughout the plant are less studied. The phloem is considered the predominant conduit for the bidirectional transport of these signals in the form of metabolites, nucleic acids, proteins, and lipids. Lipid trafficking through the phloem in particular attracted our attention due to its reliance on soluble lipid-binding proteins (LBP) that generate and solubilize otherwise membrane-associated lipids. The Phloem Lipid-Associated Family Protein (PLAFP) from Arabidopsis thaliana is generated in response to abiotic stress as is its lipid-ligand phosphatidic acid (PA). PLAFP is proposed to transport PA through the phloem in response to drought stress. To understand the interactions between PLAFP and PA, nearly 100 independent systems comprised of the protein and one PA, or a plasma membrane containing varying amounts of PA, were simulated using atomistic classical molecular dynamics methods. In these simulations, PLAFP is found to bind to plant plasma membrane models independent of the PA concentration. When bound to the membrane, PLAFP adopts a binding pose where W41 and R82 penetrate the membrane surface and anchor PLAFP. This triggers a separation of the two loop regions containing W41 and R82. Subsequent simulations indicate that PA insert into the ß-sandwich of PLAFP, driven by interactions with multiple amino acids besides the W41 and R82 identified during the insertion process. Fine-tuning the protein-membrane and protein-PA interface by mutating a selection of these amino acids may facilitate engineering plant signaling processes by modulating the binding response.

Passive permeability controls synthesis for the allelochemical sorgoleone in sorghum root exudate.

Competition for soil nutrients and water with other plants foster competition within the biosphere for access to these limited resources. The roots for the common grain sorghum produce multiple small molecules that are released via root exudates into the soil to compete with other plants. Sorgoleone is one such compound, which suppresses weed growth near sorghum by acting as a quinone analog and interferes with photosynthesis. Since sorghum also grows photosynthetically, and may be susceptible to sorgoleone action if present in tissues above ground, it is essential to exude sorgoleone efficiently. However, since the P450 enzymes that synthesize sorgoleone are intracellular, the release mechanism for sorgoleone remain unclear. In this study, we conducted an in silico assessment for sorgoleone and its precursors to passively permeate biological membranes. To facilitate accurate simulation, CHARMM parameters were newly optimized for sorgoleone and its precursors. These parameters were used to conduct 1 µs of unbiased molecular dynamics simulations to compare the permeability of sorgoleone with its precursors molecules. We find that interleaflet transfer is maximized for sorgoleone, suggesting that the precursor molecules may remain in the same leaflet for access by biosynthetic P450 enzymes. Since no sorgoleone was extracted during unbiased simulations, we compute a permeability coefficient using the inhomogeneous solubility diffusion model. The requisite free energy and diffusivity profiles for sorgoleone through a sorghum membrane model were determined through Replica Exchange Umbrella Sampling (REUS) simulations. The REUS calculations highlight that any soluble sorgoleone would quickly insert into a lipid bilayer, and would readily transit. When sorgoleone forms aggregates in root exudate as indicated by our equilibrium simulations, aggregate formation would lower the effective concentration in aqueous solution, creating a concentration gradient that would facilitate passive transport. This suggests that sorgoleone synthesis occurs within sorghum root cells and that sorgoleone is exuded by permeating through the cell membrane without the need for a transport protein once the extracellular sorgoleone aggregate is formed.

Atomistic Simulations of Polydisperse Lignin Melts Using Simple Polydisperse Residue Input Generator.

Understanding the physics of lignin will help rationalize its function in plant cell walls as well as aiding practical applications such as deriving biofuels and bioproducts. Here, we present SPRIG (Simple Polydisperse Residue Input Generator), a program for generating atomic-detail models of random polydisperse lignin copolymer melts i.e., the state most commonly found in nature. Using these models, we use all-atom molecular dynamics (MD) simulations to investigate the conformational and dynamic properties of polydisperse melts representative of switchgrass (Panicum virgatum L.) lignin. Polydispersity, branching and monolignol sequence are found to not affect the calculated glass transition temperature, Tg. The Flory-Huggins scaling parameter for the segmental radius of gyration is 0.42 ± 0.02, indicating that the chains exhibit statistics that lie between a globular chain and an ideal Gaussian chain. Below Tg the atomic mean squared displacements are independent of molecular weight. In contrast, above Tg, they decrease with increasing molecular weight. Therefore, a monodisperse lignin melt is a good approximation to this polydisperse lignin when only static properties are probed, whereas the molecular weight distribution needs to be considered while analyzing lignin dynamics.

Computing the Relative Affinity of Chlorophylls a and b to Light-Harvesting Complex II.

In plants and algae, the primary antenna protein bound to photosystem II is light-harvesting complex II (LHCII), a pigment-protein complex that binds eight chlorophyll (Chl) a molecules and six Chl b molecules. Chl a and Chl b differ only in that Chl a has a methyl group (-CH3) on one of its pyrrole rings, while Chl b has a formyl group (-CHO) at that position. This blue-shifts the Chl b absorbance relative to Chl a. It is not known how the protein selectively binds the right Chl type at each site. Knowing the selection criteria would allow the design of light-harvesting complexes that bind different Chl types, modifying an organism to utilize the light of different wavelengths. The difference in the binding affinity of Chl a and Chl b in pea and spinach LHCII was calculated using multiconformation continuum electrostatics and free energy perturbation. Both methods have identified some Chl sites where the bound Chl type (a or b) has a significantly higher affinity, especially when the protein provides a hydrogen bond for the Chl b formyl group. However, the Chl a sites often have little calculated preference for one Chl type, so they are predicted to bind a mixture of Chl a and b. The electron density of the spinach LHCII was reanalyzed, which, however, confirmed that there is negligible Chl b in the Chl a-binding sites. It is suggested that the protein chooses the correct Chl type during folding, segregating the preferred Chl to the correct binding site.

Long-Range Electron Transport Rates Depend on Wire Dimensions in Cytochrome Nanowires.

The ability to redirect electron transport to new reactions in living systems opens possibilities to store energy, generate new products, or probe physiological processes. Recent work by Huang et al. showed that 3D crystals of small tetraheme cytochromes (STC) can transport electrons over nanoscopic to mesoscopic distances by an electron hopping mechanism, making them promising materials for nanowires. However, fluctuations at room temperature may distort the nanostructure, hindering efficient electron transport. Classical molecular dynamics simulations of these fluctuations at the nano- and mesoscopic scales allowed us to develop a graph network representation to estimate maximum electron flow that can be driven through STC wires. In longer nanowires, transient structural fluctuations at protein-protein interfaces tended to obstruct efficient electron transfer, but these blockages are ameliorated in thicker crystals where alternative electron transfer pathways become more efficient. The model implies that more flexible proteinprotein interfaces limit the required minimum diameter to carry currents commensurate with conventional electronics.

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.

Nanoscale simulation of the thylakoid membrane response to extreme temperatures.

The thylakoid membrane is in a temperature-sensitive equilibrium that shifts repeatedly during the life cycle in response to ambient temperature or solar irradiance. Plants respond to seasonal temperature variation by changing their thylakoid lipid composition, while a more rapid mechanism for short-term heat exposure is required. The emission of the small organic molecule isoprene has been postulated as one such possible rapid mechanism. The protective mechanism of isoprene is unknown, but some plants emit isoprene at high temperature. We investigate the dynamics and structure for lipids within a thylakoid membrane across temperatures and varied isoprene content using classical molecular dynamics simulations. The results are compared with experimental findings for temperature-dependent changes in the lipid composition and shape of thylakoids. The surface area, volume, and flexibility of the membrane, as well as the lipid diffusion, increase with temperature, while the membrane thickness decreases. Saturated thylakoid 34:3 glycolipids derived from eukaryotic synthesis pathways exhibit altered dynamics relative to lipids from prokaryotic synthesis paths, which could explain the upregulation of specific lipid synthesis pathways at different temperatures. Increasing isoprene concentration was not observed to have a significant thermoprotective effect on the thylakoid membranes, and that isoprene readily permeated the membrane models tested.

Diffusion in intact secondary cell wall models of plants at different equilibrium moisture content.

Secondary plant cell walls are composed of carbohydrate and lignin polymers, and collectively represent a significant renewable resource. Leveraging these resources depends in part on a mechanistic understanding for diffusive processes within plant cell walls. Common wood protection treatments and biomass conversion processes to create biorefinery feedstocks feature ion or solvent diffusion within the cell wall. X-ray fluorescence microscopy experiments have determined that ionic diffusion rates are dependent on cell wall hydration as well as the ionic species through non-linear relationships. In this work, we use classical molecular dynamics simulations to map the diffusion behavior of different plant cell wall components (cellulose, hemicellulose, lignin), ions (Na+, K+, Cu2+, Cl-) and water within a model for an intact plant cell wall at various hydration states (3-30 wt% water). From these simulations, we analyze the contacts between different plant cell wall components with each other and their interaction with the ions. Generally, diffusion increases with increasing hydration, with lignin and hemicellulose components increasing diffusion by an order of magnitude over the tested hydration range. Ion diffusion depends on charge. Positively charged cations preferentially interact with hemicellulose components, which include negatively charged carboxylates. As a result, positive ions diffuse more slowly than negatively charged ions. Measured diffusion coefficients are largely observed to best fit piecewise linear trends, with an inflection point between 10 and 15% hydration. These observations shed light onto the molecular mechanisms for diffusive processes within secondary plant cell walls at atomic resolution.

SARS-CoV2 billion-compound docking.

This dataset contains ligand conformations and docking scores for 1.4 billion molecules docked against 6 structural targets from SARS-CoV2, representing 5 unique proteins: MPro, NSP15, PLPro, RDRP, and the Spike protein. Docking was carried out using the AutoDock-GPU platform on the Summit supercomputer and Google Cloud. The docking procedure employed the Solis Wets search method to generate 20 independent ligand binding poses per compound. Each compound geometry was scored using the AutoDock free energy estimate, and rescored using RFScore v3 and DUD-E machine-learned rescoring models. Input protein structures are included, suitable for use by AutoDock-GPU and other docking programs. As the result of an exceptionally large docking campaign, this dataset represents a valuable resource for discovering trends across small molecule and protein binding sites, training AI models, and comparing to inhibitor compounds targeting SARS-CoV-2. The work also gives an example of how to organize and process data from ultra-large docking screens.

Characterization of promoter elements of isoprene-responsive genes and the ability of isoprene to bind START domain transcription factors.

Isoprene has recently been proposed to be a signaling molecule that can enhance tolerance of both biotic and abiotic stress. Not all plants make isoprene, but all plants tested to date respond to isoprene. We hypothesized that isoprene interacts with existing signaling pathways rather than requiring novel mechanisms for its effect on plants. We analyzed the cis-regulatory elements (CREs) in promoters of isoprene-responsive genes and the corresponding transcription factors binding these promoter elements to obtain clues about the transcription factors and other proteins involved in isoprene signaling. Promoter regions of isoprene-responsive genes were characterized using the Arabidopsis cis-regulatory element database. CREs bind ARR1, Dof, DPBF, bHLH112, GATA factors, GT-1, MYB, and WRKY transcription factors, and light-responsive elements were overrepresented in promoters of isoprene-responsive genes; CBF-, HSF-, WUS-binding motifs were underrepresented. Transcription factors corresponding to CREs overrepresented in promoters of isoprene-responsive genes were mainly those important for stress responses: drought-, salt/osmotic-, oxidative-, herbivory/wounding and pathogen-stress. More than half of the isoprene-responsive genes contained at least one binding site for TFs of the class IV (homeodomain leucine zipper) HD-ZIP family, such as GL2, ATML1, PDF2, HDG11, ATHB17. While the HD-zipper-loop-zipper (ZLZ) domain binds to the L1 box of the promoter region, a special domain called the steroidogenic acute regulatory protein-related lipid transfer, or START domain, can bind ligands such as fatty acids (e.g., linolenic and linoleic acid). We tested whether isoprene might bind in such a START domain. Molecular simulations and modeling to test interactions between isoprene and a class IV HD-ZIP family START-domain-containing protein were carried out. Without membrane penetration by the HDG11 START domain, isoprene within the lipid bilayer was inaccessible to this domain, preventing protein interactions with membrane bound isoprene. The cross-talk between isoprene-mediated signaling and other growth regulator and stress signaling pathways, in terms of common CREs and transcription factors could enhance the stability of the isoprene emission trait when it evolves in a plant but so far it has not been possible to say what how isoprene is sensed to initiate signaling responses.

LongBondEliminator: A Molecular Simulation Tool to Remove Ring Penetrations in Biomolecular Simulation Systems.

We develop a workflow, implemented as a plugin to the molecular visualization program VMD, that can fix ring penetrations with minimal user input. LongBondEliminator, detects ring piercing artifacts by the long, strained bonds that are the local minimum energy conformation during minimization for some assembled simulation system. The LongBondEliminator tool then automatically treats regions near these long bonds using multiple biases applied through NAMD. By combining biases implemented through the collective variables module, density-based forces, and alchemical techniques in NAMD, LongBondEliminator will iteratively alleviate long bonds found within molecular simulation systems. Through three concrete examples with increasing complexity, a lignin polymer, an viral capsid assembly, and a large, highly glycosylated protein aggrecan, we demonstrate the utility for this method in eliminating ring penetrations from classical MD simulation systems. The tool is available via gitlab as a VMD plugin, and has been developed to be generically useful across a variety of biomolecular simulations.

Plant Terpenoid Permeability through Biological Membranes Explored via Molecular Simulations.

Plants synthesize small molecule diterpenes composed of 20 carbons from precursor isopentenyl diphosphate and dimethylallyl disphosphate, manufacturing diverse compounds used for defense, signaling, and other functions. Industrially, diterpenes are used as natural aromas and flavoring, as pharmaceuticals, and as natural insecticides or repellents. Despite diterpene ubiquity in plant systems, it remains unknown how plants control diterpene localization and transport. For many other small molecules, plant cells maintain transport proteins that control compound compartmentalization. However, for most diterpene compounds, specific transport proteins have not been identified, and so it has been hypothesized that diterpenes may cross biological membranes passively. Through molecular simulation, we study membrane transport for three complex diterpenes from among the many made by members of the Lamiaceae family to determine their permeability coefficient across plasma membrane models. To facilitate accurate simulation, the intermolecular interactions for leubethanol, abietic acid, and sclareol were parametrized through the standard CHARMM methodology for incorporation into molecular simulations. To evaluate the effect of membrane composition on permeability, we simulate the three diterpenes in two membrane models derived from sorghum and yeast lipidomics data. We track permeation events within our unbiased simulations, and compare implied permeation coefficients with those calculated from Replica Exchange Umbrella Sampling calculations using the inhomogeneous solubility diffusion model. The diterpenes are observed to permeate freely through these membranes, indicating that a transport protein may not be needed to export these small molecules from plant cells. Moreover, the permeability is observed to be greater for plant-like membrane compositions when compared against animal-like membrane models. Increased permeability for diterpene molecules in plant membranes suggest that plants have tailored their membranes to facilitate low-energy transport processes for signaling molecules.

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface.

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

Molecular simulation of lignin-related aromatic compound permeation through gram-negative bacterial outer membranes.

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.

Reversible Unwrapping Algorithm for Constant-Pressure Molecular Dynamics Simulations.

Molecular simulation technologies have afforded researchers a unique look into the nanoscale interactions driving physical processes. However, a limitation for molecular dynamics (MD) simulations is that they must be performed on finite-sized systems in order to map onto computational resources. To minimize artifacts arising from finite-sized simulation systems, it is common practice for MD simulations to be performed with periodic boundary conditions (PBCs). However, in order to calculate specific physical properties, such as mean square displacements to calculate diffusion coefficients, continuous particle trajectories where the atomic movements are continuous and do not jump between cell faces are required. In these cases, modifying atomic coordinates through unwrapping schemes is an essential post-processing tool to remove these jumps. Here, two established trajectory unwrapping schemes are applied to 1 µs wrapped trajectories for a small water box and lysozyme in water. The existing schemes can result in spurious diffusion coefficients, long bonds within unwrapped molecules, and inconsistent atomic coordinates when coordinates are rewrapped after unwrapping. We determine that prior unwrapping schemes do not account for changing periodic box dimensions and introduce an additional correction term to the existing displacement unwrapping scheme to correct for these artifacts. We also demonstrate that the resulting algorithm is a hybrid between the existing heuristic and displacement unwrapping schemes. After treatment using this new unwrapping scheme, molecular geometries are correct even after long simulations. In anticipation for longer MD trajectories, we develop implementations for this new scheme in multiple PBC handling tools.

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.

Assembly and Analysis of Cell-Scale Membrane Envelopes.

The march toward exascale computing will enable routine molecular simulation of larger and more complex systems, for example, simulation of entire viral particles, on the scale of approximately billions of atoms─a simulation size commensurate with a small bacterial cell. Anticipating the future hardware capabilities that will enable this type of research and paralleling advances in experimental structural biology, efforts are currently underway to develop software tools, procedures, and workflows for constructing cell-scale structures. Herein, we describe our efforts in developing and implementing an efficient and robust workflow for construction of cell-scale membrane envelopes and embedding membrane proteins into them. A new approach for construction of massive membrane structures that are stable during the simulations is built on implementing a subtractive assembly technique coupled with the development of a structure concatenation tool (fastmerge), which eliminates overlapping elements based on volumetric criteria rather than adding successive molecules to the simulation system. Using this approach, we have constructed two "protocells" consisting of MARTINI coarse-grained beads to represent cellular membranes, one the size of a cellular organelle and another the size of a small bacterial cell. The membrane envelopes constructed here remain whole during the molecular dynamics simulations performed and exhibit water flux only through specific proteins, demonstrating the success of our methodology in creating tight cell-like membrane compartments. Extended simulations of these cell-scale structures highlight the propensity for nonspecific interactions between adjacent membrane proteins leading to the formation of protein microclusters on the cell surface, an insight uniquely enabled by the scale of the simulations. We anticipate that the experiences and best practices presented here will form the basis for the next generation of cell-scale models, which will begin to address the addition of soluble proteins, nucleic acids, and small molecules essential to the function of a cell.