Model building into experimental maps is a key element of structural biology, but can be both time consuming and error prone for low-resolution maps. Here we present Namdinator, an easy-to-use tool that enables the user to run a molecular dynamics flexible fitting simulation followed by real-space refinement in an automated manner through a pipeline system. Namdinator will modify an atomic model to fit within cryo-EM or crystallography density maps, and can be used advantageously for both the initial fitting of models, and for a geometrical optimization step to correct outliers, clashes and other model problems. We have benchmarked Namdinator against 39 deposited cryo-EM models and maps, and observe model improvements in 34 of these cases (87%). Clashes between atoms were reduced, and the model-to-map fit and overall model geometry were improved, in several cases substantially. We show that Namdinator is able to model large-scale conformational changes compared to the starting model. Namdinator is a fast and easy tool for structural model builders at all skill levels. Namdinator is available as a web service (https://namdinator.au.dk), or it can be run locally as a command-line tool.

Bioelectricity generation, by Shewanella oneidensis (S. oneidensis) MR-1, has become particularly alluring, thanks to its extraordinary prospects for energy production, pollution treatment, and biosynthesis. Attempts to improve its technological output by modification of S. oneidensis MR-1 remains complicated, expensive and inefficient. Herein, we report on the augmentation of S. oneidensis MR-1 with carbon dots (CDs). The CDs-fed cells show accelerated extracellular electron transfer and metabolic rate, with increased intracellular charge, higher adenosine triphosphate level, quicker substrate consumption and more abundant extracellular secretion. Meanwhile, the CDs promote cellular adhesion, electronegativity, and biofilm formation. In bioelectrical systems the CDs-fed cells increase the maximum current value, 7.34 fold, and power output, 6.46 fold. The enhancement efficacy is found to be strongly dependent on the surface charge of the CDs. This work demonstrates a simple, cost-effective and efficient route to improve bioelectricity generation of S. oneidensis MR-1, holding promise in all relevant technologies.

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae. Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation.

Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the β-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.