NAMD is a molecular dynamics program designed for high-performance simulations of very large biological objects on central processing unit (CPU)-and graphics processing unit (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, as well as hybrid QM/MM 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 towards achieving optimal performance on GPUbased 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.

SUMMARY The transduction of transmembrane electric fields into protein motion plays an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSD) carry out these functions through reorientations of S4 helix with discrete gating charges. Here, crystal structures of the VSD from Ci-VSP were determined in both, active (Up) and resting (Down) conformations. The S4 undergoes a ~5 Å displacement along its main axis accompanied by a ~60o rotation, consistent with the helix-screw gating mechanism. This movement is stabilized by a change in countercharge partners in helices S1 and S3, generating an estimated net charge transfer of ~1 eo. Gating charges move relative to a “hydrophobic gasket” that electrically divides intra and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent cellular activities.

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.DOI: http://dx.doi.org/10.7554/eLife.16105.001

Order parameters (OPs) characterizing the nanoscale features of macromolecules are presented. They are generated in a general fashion so that they do not need to be redesigned with each new application. They evolve on time scales much longer than 10 −14 s typical for individual atomic collisions/vibrations. The list of OPs can be automatically increased, and completeness can be determined via a correlation analysis. They serve as the basis of a multiscale analysis that starts with the N-atom Liouville equation and yields rigorous Smoluchowski/Langevin equations of stochastic OP dynamics. Such OPs and the multiscale analysis imply computational algorithms that we demonstrate in an application to ribonucleic acid structural dynamics for 50 ns.

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-lightadapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc 1 complex (cytbc 1 ) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%-5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.transition metals | MntH | divalent metal transporter DMT1 | hard-soft acid-base theory | ion selectivity filters A ll organisms require transition metal ions as cofactors in proteins that perform a variety of essential cellular tasks. Through evolution, organisms have developed mechanisms to acquire, transport, and safely store essential metals such as manganese, iron, cobalt, and zinc. The natural resistance-associated macrophage protein (Nramp) family of metal transporters represents a common transition metal acquisition strategy conserved across all kingdoms of life (1). The first discovered mammalian Nramp (Nramp1) is expressed in phagosomal membranes and likely extracts essential metals to help kill engulfed pathogens (2, 3). Mammals use Nramp2, an essential gene also called DMT1, to absorb dietary iron into the enterocytes that line the small intestine (4) and to extract iron from transferrin-containing endosomes in all tissues. Bacteria express their own Nramp homologs, which they typically use to scavenge manganese and other first row divalent transition metals (5, 6). Last, most plants have several Nramp homologs that take up iron and manganese, the essential cofactor in photosystem II, from the soil or vacuolar stores (7,8).Nramps are generally thought to function as metal-proton symporters (1) and are able to bind and/or transport a wide range of divalent transition metal substrates, including the biologically useful metals Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , as well as the toxic heavy metals Cd 2+ , Pb 2+ , and Hg 2+ (4, 9-13). Nramps do discriminate against the divalent alkaline earth metal ions Mg 2+ and Ca 2+ (9, 14), which are typically several orders of magnitude mor...

Summary The widely-conserved natural resistance associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a—absent from a previous Nramp structure. Mapping our cysteine accessibility scanning results onto this structure, we identified the metal permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations, which impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.